Neuregulin variants and methods of screening and using thereof

ABSTRACT

The present invention provides polypeptide variants of neuregulin-1β (NRG-1β) that have enhanced or decreased binding affinity to ErbB3 and/or ErbB4. The invention also provides methods of screening and producing polypeptide variants of NRG-1β and methods of using polypeptide variants of NRG-1β for treating diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims the prioritybenefit of U.S. patent application Ser. No. 11/293,879 filed Dec. 2,2005, which is incorporated herein by references in its entirety.

FIELD OF THE INVENTION

This invention relates generally to neuregulin variants that selectivelyactivate ErbB receptors. The invention provides neuregulin variants andmethods for screening and using such variants.

BACKGROUND OF THE INVENTION

The epidermal growth factor receptor family, which comprises fourmembers EGFR, ErbB2, ErbB3 and ErbB4, has been demonstrated to play animportant role in multiple cellular functions, including cell growth,differentiation and survival. They are protein tyrosine kinasereceptors, consisting of an extracellular ligand-binding domain,transmembrane domain and cytoplasmic tyrosine kinase domain. Multiplereceptor ligands have been identified which mediate receptor homo- orhetero-dimerization upon binding. The specific receptor associationresults in different patterns of phosphorylation, complex signalingcascades and multiple biological functions, including cellularproliferation, prevention of apoptosis and promotion of tumor cellmobility, adhesion and invasion.

Neuregulin-1 is a ligand of ErbB3 and ErbB4 receptors. Over 15 distinctisoforms of neuregulin-1 have been identified. Neuregulin-1 isoforms canbe divided into two large groups, known as α- and β-types, on the basisof differences in the structure of their essential epidermal growthfactor (EGF)-like domains. It has been shown that the EGF-like domainsof neuregulin-1, ranging in size from 50 to 64-amino acids, aresufficient to bind to and activate these receptors. Previous studieshave shown that neuregulin-1β (NRG-1β) can bind directly to ErbB3 andErbB4 with high affinity. The orphan receptor, ErbB2, holds apre-activated conformation to facilitate hetero-dimerization with ErbB3or ErbB4 with approximately 100-fold higher affinity than ErbB3 andErbB4 homodimers. The heterometric receptors act in distinct cell types:ErbB2/ErbB3 in the peripheral nervous system and ErbB2/ErbB4 in theheart. Research in neural development has indicated that the formationof the sympathetic nervous system requires an intact NRG-1β, ErbB2 andErbB3 signaling system. Targeted disruption of the NRG-1β or ErbB2 orErbB4 led to embryonic lethality due to cardiac development defects.Recent studies also highlighted the roles of NRG-1β, ErbB2 and ErbB4 inthe cardiovascular development as well as in the maintenance of adultnormal heart function. NRG-1β has been shown to enhance sarcomereorganization in adult cardiomyocytes. The short-term administration of arecombinant NRG-1β EGF domain significantly improves or protects againstdeterioration in myocardial performance in three distinct animal modelsof heart failure. More importantly, NRG-1β significantly prolongssurvival of heart failure animals. These effects make NRG-1β promisingas a broad spectrum therapeutic or lead compound for heart failure dueto a variety of common diseases. However, there is still a need fordetailed structural information of NRG-1β in complex with its receptorsfor designing variants of NRG-1β for therapeutic use.

Numerous computational studies employing homology modeling, moleculardynamics simulations and free energy calculations have been carried outfor ligand-protein and protein-protein interactions at the atomic level.Prediction of absolute ligand-receptor binding free energies isessential in a wide range of biophysical queries such as structure-baseddrug design. Recently, a new computational approach, the MolecularMechanics Poisson Boltzmann Surface Area (MM-PBSA), has been used forstudying protein-protein interactions. MM-PBSA calculates the freeenergies of the end states directly to avoid the time-consumingsimulation of the intermediate states. This method combines molecularmechanical energies for the solute with a continuum solvent approach andnormal mode analysis to estimate the total free energies. Computationalalanine-scanning methodology has also been used for studyingprotein-protein interactions.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a polypeptide variant ofneuregulin-1β comprising amino acid sequence shown in SEQ ID NO:1,wherein the polypeptide variant comprises a different amino acid thanthat in SEQ ID NO:1, wherein the polypeptide variant has an enhancedbinding affinity to ErbB3 compared to polypeptide of SEQ ID NO:1, andwherein at residue 25 said different amino acid is A, C, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W, or Y; at residue 35 said differentamino acid is A, C, D, E, F, G, H, I, L, N, M, P, Q, R, S, T, V, W, orY; and/or at residue 46 said different amino acid is A, C, D, E, F, G,H, I, K, L, M, N, P, R, S, T, V, W, or Y.

In some embodiments, the polypeptide variant consists of the amino acidsequence shown in SEQ ID NO:1, and wherein at residue 25 said differentamino acid is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; at residue 35 said different amino acid is A, C, D, E, F, G, H, I, L,N, M, P, Q, R, S, T, V, W, or Y; and/or at residue 46 said differentamino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, orY.

In some embodiments of the polypeptide variants, at residue 25 saiddifferent amino acid is A; at residue 35 said different amino acid is A;and/or at residue 46 said different amino acid is A.

In some embodiments, the polypeptide variant has a decreased or similarbinding affinity to ErbB4 compared to the polypeptide of SEQ ID NO:1. Insome embodiments of the polypeptide variants, at residue 25 saiddifferent amino acid is A. In some embodiments of the polypeptidevariants, at residue 35 said different amino acid is A. In someembodiments of the polypeptide variants, at residue 46 said differentamino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βcomprised of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has an enhanced binding affinityto ErbB3 compared to polypeptide consisting of amino acid residues 1-52of SEQ ID NO:1, and wherein at residue 25 said different amino acid isA, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; at residue35 said different amino acid is A, C, D, E, F, G, H, I, L, N, M, P, Q,R, S, T, V, W, or Y; and/or at residue 46 said different amino acid isA, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y.

In some embodiments of the polypeptide variants, at residue 25 saiddifferent amino acid is A; at residue 35 said different amino acid is A;and/or at residue 46 said different amino acid is A.

In some embodiments, the polypeptide variant has a decreased or similarbinding affinity to an ErbB4 compared to the polypeptide consisting ofamino acid residues 1-52 of SEQ ID NO:1. In some embodiments of thepolypeptide variants, at residue 25 said different amino acid is A. Insome embodiments of the polypeptide variants, at residue 35 saiddifferent amino acid is A. In some embodiments of the polypeptidevariants, at residue 46 said different amino acid is A.

The invention also provides a polynucleotide comprising a nucleic acidsequence encoding the polypeptide variant described herein that has anenhanced binding affinity to ErbB3 compared to polypeptide of SEQ IDNO:1 or polypeptide consisting of amino acid residues 1-52 of SEQ INNO:1.

The invention also provides a pharmaceutical composition comprising aneffective amount of the polypeptide variant described herein that has anenhanced binding affinity to ErbB3 compared to polypeptide of SEQ IDNO:1 or polypeptide consisting of amino acid residues 1-52 of SEQ INNO:1, or the polynucleotide encoding the polypeptide variant and apharmaceutically acceptable excipient.

The invention also provides a kit comprising the pharmaceuticalcomposition. In some embodiments, the kit further comprises aninstruction for using the pharmaceutical composition in preventing,treating, or delaying a disease in an individual via activatingErbB2/ErbB3 receptors.

The invention also provides a method for preventing, treating, ordelaying development of schizophrenia in a mammal, comprisingadministering to a mammal, to which such prevention, treatment or delayis needed or desirable, a pharmaceutical composition comprising aneffective amount of the pharmaceutical composition. In some embodiments,the mammal is a human.

In another aspect, the present invention provides a polypeptide variantof neuregulin-1β comprising amino acid sequence shown in SEQ ID NO:1,wherein the polypeptide variant comprises a different amino acid thanthat in SEQ ID NO:1, wherein the polypeptide variant has a decreasedbinding affinity to ErbB4 compared to polypeptide of SEQ ID NO:1, andwherein at residue 3 said different amino acid is A, C, D, E, F, G, H,I, K, M, N, P, Q, R, S, T, V, W, or Y.

In some embodiments, the polypeptide variant consists of the amino acidsequence shown in SEQ ID NO:1, and wherein at residue 3 said differentamino acid is A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY.

In some embodiments of the polypeptide variants, at residue 3 saiddifferent amino acid is A.

In some embodiments, the polypeptide variant has an increased or similarbinding affinity to ErbB3 compared to the polypeptide of SEQ ID NO:1. Insome embodiments of the polypeptide variants, at residue 3 saiddifferent amino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βcomprised of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ.ID NO:1, wherein the polypeptide variant has a decreased bindingaffinity to ErbB4 compared to polypeptide consisting of amino acidresidues 1-52 of SEQ ID NO:1, and wherein at residue 3 said differentamino acid is A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY.

In some embodiments of the polypeptide variants, at residue 3 saiddifferent amino acid is A.

In some embodiments, the polypeptide variant has an enhanced or similarbinding affinity to an ErbB3 compared to the polypeptide consisting ofamino acid residues 1-52 of SEQ ID NO:1. In some embodiments of thepolypeptide variants, at residue 3 said different amino acid is A.

The invention also provides a polynucleotide comprising a nucleic acidsequence encoding the polypeptide variant described herein that has adecreased binding affinity to ErbB4 compared to polypeptide of SEQ IDNO:1 or polypeptide consisting of amino acid residues 1-52 of SEQ IDNO:1.

The invention also provides a pharmaceutical composition comprising aneffective amount of the polypeptide variant described herein that has adecreased binding affinity to ErbB4 compared to polypeptide of SEQ IDNO:1 or polypeptide consisting of amino acid residues 1-52 of SEQ INNO:1, or the polynucleotide encoding the polypeptide variant and apharmaceutically acceptable excipient.

The invention also provides a kit comprising the pharmaceuticalcomposition. In some embodiments, the kit further comprises aninstruction for using the pharmaceutical composition in preventing,treating, or delaying a disease in an individual via activatingErbB2/ErbB3.

The invention also provides a method for preventing, treating, ordelaying development of schizophrenia in a mammal, comprisingadministering to a mammal, to which such prevention, treatment or delayis needed or desirable, a pharmaceutical composition comprising aneffective amount of the pharmaceutical composition. In some embodiments,the mammal is a human.

In another aspect, the present invention provides a polypeptide variantof neuregulin-1β comprising amino acid sequence shown in SEQ ID NO:1,wherein the polypeptide variant comprises a different amino acid thanthat in SEQ ID NO:1, wherein the polypeptide variant has an enhancedbinding affinity to ErbB4 compared to polypeptide of SEQ ID NO:1, andwherein at residue 16 said different amino acid is A, C, D, E, F, G, H,I, K, L, M, P, Q, R, S, T, V, W, or Y; at residue 29 said differentamino acid is A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; at residue 31 said different amino acid is A, C, D, E, F, G, H, I, K,L, M, N, P, Q, S, T, V, W, or Y; at residue 43 said different amino acidis A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and/or atresidue 47 said different amino acid is A, C, D, E, F, G, H, I, K, L, M,P, Q, R, S, T, V, W, or Y.

In some embodiments, the polypeptide variant consists of the amino acidsequence shown in SEQ ID NO:1, and wherein at residue 16 said differentamino acid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; at residue 29 said different amino acid is A, C, D, E, F, G, H, I, K,L, M, N, Q, R, S, T, V, W, or Y; at residue 31 said different amino acidis A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; atresidue 43 said different amino acid is A, C, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; and/or at residue 47 said different aminoacid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y.

In some embodiments of the polypeptide variants, at residue 16 saiddifferent amino acid is A; at residue 29 said different amino acid is A;at residue 31 said different amino acid is A; at residue 43 saiddifferent amino acid is A; or at residue 47 said different ammo acid isA.

In some embodiments, the polypeptide variant has a decreased or similarbinding affinity to an ErbB3 compared to the polypeptide of SEQ ID NO:1.In some embodiments of the polypeptide variants, at residue 31 saiddifferent amino acid is A. In some embodiments of the polypeptidevariants, at residue 43 said different amino acid is A. In someembodiments of the polypeptide variants, at residue 47 said differentamino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βconsisting of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has an enhanced, binding affinityto ErbB4 compared to polypeptide consisting of amino acid residues 1-52of SEQ ID NO:1, and wherein at residue 16 said different amino acid isA, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; at residue29 said different amino acid is A, C, D, E, F, G, H, I, K, L, M, N, Q,R, S, T, V, W, or Y; at residue 31 said different amino acid is A, C, D,B, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; at residue 43 saiddifferent amino acid is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, W, or Y; and/or at residue 47 said different amino acid is A, C, D,E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y.

In some embodiments of the polypeptide variants, at residue 16 saiddifferent amino acid is A; at residue 29 said different amino acid is A;at residue 31 said different amino acid is A; at residue 43 saiddifferent amino acid is A; and/or at residue 47 said different aminoacid is A.

In some embodiments, the polypeptide variant has a decreased or similarbinding affinity to an ErbB3 compared to the polypeptide of SEQ ID NO:1.In some embodiments of the polypeptide variants, at residue 31 saiddifferent amino acid is A. In some embodiments of the polypeptidevariants, at residue 43 said different amino acid is A. In someembodiments of the polypeptide variants, at residue 47 said differentamino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βcomprising amino acid sequence of SEQ ID NO:1, wherein the polypeptidevariant comprises a different amino acid than that in SEQ ID NO:1,wherein the polypeptide variant has a decreased binding affinity toErbB3 compared to polypeptide of SEQ ID NO:1 but has a binding affinityto ErbB4 similar to polypeptide of SEQ ID NO:1, and wherein at residue33 said different amino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βconsisting of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has a decreased binding affinityto ErbB3 compared to polypeptide consisting of amino acid residues 1-52of SEQ ID NO:1 but has a binding affinity to ErbB4 similar topolypeptide consisting of amino acid residues 1-52 SEQ ID NO:1, andwherein at residue 33 said different amino acid is A.

The invention also provides a polynucleotide comprising a nucleic acidsequence encoding the polypeptide variant described herein that has anenhanced binding affinity to ErbB4 compared to polypeptide of SEQ IDNO:1 or polypeptide consisting of amino acid residues 1-52 of SEQ IDNO:1.

The invention also provides a pharmaceutical composition comprising aneffective amount of the polypeptide variant described herein that has anenhanced binding affinity to ErbB4 compared to polypeptide of SEQ IDNO:1 or polypeptide consisting of amino acid residues 1-52 of SEQ IDNO:1, or the polynucleotide encoding the polypeptide variant and apharmaceutically acceptable excipient.

The invention also provides a kit comprising the pharmaceuticalcomposition. In some embodiments, the kit further comprises aninstruction for using the pharmaceutical composition in preventing,treating, or delaying a disease in an individual via activatingErbB2/ErbB4 receptors.

The invention also provides a method for preventing, treating, ordelaying development of viral myocarditis, dilated (congestive)cardiomyopathy, cardiac toxicity, heart failure, myocardial infarctionin an individual, comprising administering to an individual, to whichsuch prevention, treatment or delay is needed or desirable, apharmaceutical composition comprising an effective amount of thepharmaceutical composition. In some embodiments, the mammal is a human.

In another aspect, the invention provides a method for screening apolypeptide variant of neuregulin-1β having enhanced binding affinity toErbB3, which method comprises: (a) establishing a three-dimensionalstructure of a neuregulin-1β or a fragment thereof, an ErbB3, and acomplex of the neuregulin-1β or the fragment thereof and the ErbB3 byhomology modeling; (b) establishing data of conformational changes andstability of the complex of the neuregulin-1β or the fragment thereofand the ErbB3 in solution by molecular dynamics simulation method; (c)calculating subtotal binding free energy (ΔG_(subtotal wildtype)) of theneuregulin-1β or the fragment thereof with the ErbB3 by MolecularMechanics Poisson Boltzmann Surface Area (MM-PBSA) method; (d)calculating subtotal binding free energy(ΔG_(subtotal alanine substituted variant)) of an alanine substitutedvariant of the neuregulin-1β or the fragment thereof with the ErbB3 byMolecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method,wherein the alanine substituted variant comprises an amino acid of theneuregulin-1β or the fragment thereof substituted, by an alanine; (e)calculatingΔΔG_(subtotal)=ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);and (f) selecting alanine substituted variant that has a positive valueof ΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3; whereby a polypeptide variant of neuregulin-1βthat has enhanced binding affinity to ErbB3 is identified.

The invention also provides a method for screening a polypeptide variantof neuregulin-1β having enhanced binding affinity selective to ErbB3,which method comprises: (a) establishing a three-dimensional structureof a neuregulin-1β or a fragment thereof, an ErbB3, an ErbB4, a complexof the neuregulin-1β or the fragment thereof and the ErbB3, and acomplex of the neuregulin-1β or the fragment thereof and the ErbB4 byhomology modeling; (b) establishing data of conformational changes andstability of the complex of the neuregulin-1β or the fragment thereofand the ErbB3, and the complex of the neuregulin-1β or the fragmentthereof and the ErbB4 in solution by molecular dynamics simulationmethod; (c) calculating subtotal binding free energy(ΔG_(subtotal wildtype)) of the neuregulin-1β or the fragment thereofwith the ErbB3 or the ErbB4 by Molecular Mechanics Poisson BoltzmannSurface Area (MM-PBSA) method; (d) calculating subtotal binding freeenergy (ΔG_(subtotal alanine substituted variant)) of an alaninesubstituted variant of the neuregulin-1β or the fragment thereof withthe ErbB3 or the ErbB4 by Molecular Mechanics Poisson Boltzmann SurfaceArea (MM-PBSA) method, wherein the alanine substituted variant comprisesan amino acid of the neuregulin-1β or the fragment thereof substitutedby an alanine; (e) calculatingΔΔG_(subtotal)=ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);(f) selecting alanine substituted variant that has a positive value ofΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3, and has a negative value or a value of about zerofor ΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB4; whereby a polypeptide variant of neuregulin-1βthat has enhanced binding affinity selective to ErbB3 is identified. Insome embodiments, an alanine substituted variant having the negativevalue for ΔΔG_(subtotal) for the complex of the neuregulin-1β or thefragment thereof and the ErbB4 is selected, whereby a polypeptidevariant of neuregulin-1β that has enhanced binding affinity to ErbB3 butdecreased binding affinity to ErbB4 is identified. In some embodiments,an alanine substituted variant having the value of about zero forΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB4 is selected, whereby a polypeptide variant ofneuregulin-1β that has enhanced binding affinity to ErbB3 but unchangedbinding affinity to ErbB4 is identified.

In another aspect, the invention provides a method for screening, apolypeptide variant of neuregulin-1β having enhanced binding affinity toErbB4, which method comprises: (a) establishing a three-dimensionalstructure of a neuregulin-1β or a fragment thereof, an ErbB4, and acomplex of the neuregulin-1β or the fragment thereof and the ErbB4 byhomology modeling; (b) establishing data of conformational changes andstability of the complex of the neuregulin-1β or the fragment thereofand the ErbB4 in solution by molecular dynamics simulation method; (c)calculating subtotal binding free energy (ΔG_(subtotal wildtype)) of theneuregulin-1β or the fragment thereof with the ErbB4 by MolecularMechanics Poisson Boltzmann Surface Area (MM-PBSA) method; (d)calculating subtotal binding free energy(<G_(subtotal alanine substituted variant)) of an alanine substitutedvariant of the neuregulin-1β or the fragment thereof with the ErbB4 byMolecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method,wherein the alanine substituted variant comprises an amino acid of theneuregulin-1β or the fragment thereof substituted by an alanine; (e)calculatingΔΔG_(subtotal)=ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);and (f) selecting an alanine substituted variant that has a positivevalue of ΔΔG_(subtotal) for the complex of the neuregulin-1β or thefragment thereof and the ErbB4; whereby a polypeptide variant ofneuregulin-1β that has enhanced binding affinity to ErbB4 is identified.

The invention also provides a method for screening a polypeptide variantof neuregulin-1β having enhanced binding affinity selective to ErbB4,which method comprises: (a) establishing a three-dimensional structureof a neuregulin-1β or a fragment thereof, an ErbB3, an ErbB4, a complexof the neuregulin-1β or the fragment thereof and the ErbB3, and acomplex of the neuregulin-1β or the fragment thereof and the ErbB4 byhomology modeling; (b) establishing data of conformational changes andstability of the complex of the neuregulin-1β or the fragment thereofand the ErbB3, and the complex of the neuregulin-1β or the fragmentthereof and the ErbB4 in solution by molecular dynamics simulationmethod; (c) calculating subtotal binding free energy(ΔG_(subtotal wildtype)) of the neuregulin-1β or the fragment thereofwith the ErbB3 or the ErbB4 by Molecular Mechanics Poisson BoltzmannSurface Area (MM-PB SA) method; (d) calculating subtotal binding freeenergy (ΔG_(subtotal alanine substituted variant)) of an alaninesubstituted variant of the neuregulin-1β or the fragment thereof withthe ErbB3 or the ErbB4 by Molecular Mechanics Poisson Boltzmann SurfaceArea (MM-PBSA) method, wherein the alanine substituted variant comprisesan amino acid of the neuregulin-1β or the fragment thereof substitutedby an alanine; (e) calculatingΔΔG_(subtotal)=ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);(f) selecting alanine substituted variant that has a positive value ofΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB4, and has a negative value or a value of about zerofor ΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3; whereby a polypeptide variant of neuregulin-1βthat has enhanced binding affinity selective to ErbB4 is identified. Insome embodiments, alanine substituted variant having the negative valuefor ΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3 is selected, whereby a polypeptide variant ofneuregulin-1β that has enhanced binding affinity to ErbB4 but decreasedbinding affinity to ErbB3 is identified. In some embodiments, alaninesubstituted variant having the value of about zero for ΔΔG_(subtotal)for the complex of the neuregulin-1β or the fragment thereof and theErbB3 is selected, whereby a polypeptide variant of neuregulin-1β thathas enhanced binding affinity to ErbB4 but unchanged binding affinity toErbB3 is identified.

The invention also provides a method for screening a polypeptide variantof neuregulin-1β having unchanged binding affinity to ErbB4 but hasdecreased binding affinity to ErbB3, which method comprises: (a)establishing a three-dimensional structure of a neuregulin-1β or afragment thereof, an ErbB3, an ErbB4, a complex of the neuregulin-1β orthe fragment thereof and the ErbB3, and a complex of the neuregulin-1βor the fragment thereof and the ErbB4 by homology modeling; (b)establishing data of conformational changes and stability of the complexof the neuregulin-1β or the fragment thereof and the ErbB3, and thecomplex of the neuregulin-1β or the fragment thereof and the ErbB4 insolution by molecular dynamics simulation method; (c) calculatingsubtotal binding free energy (ΔG_(subtotal wildtype)) of theneuregulin-1β or the fragment thereof with the ErbB3 or the ErbB4 byMolecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method; (d)calculating subtotal binding free energy(ΔG_(subtotal alanine substituted variant)) of an alanine substitutedvariant of the neuregulin-1β or the fragment thereof with the ErbB3 orthe ErbB4 by Molecular Mechanics Poisson Boltzmann Surface Area(MM-PBSA) method, wherein the alanine substituted variant comprises anamino acid of the neuregulin-1β or the fragment thereof substituted byan alanine; (e) calculatingΔΔG_(subtotal)×ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);(f) selecting alanine substituted variant that has a value of about zerofor ΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB4 and has a negative value for ΔΔG_(subtotal) forthe complex of the neuregulin-1β or the fragment thereof and the ErbB3;whereby a polypeptide variant of neuregulin-1β that has unchangedbinding affinity to ErbB4 but has decreased binding affinity to ErbB3 isidentified.

The invention also provides a polypeptide variant of a neuregulin-1βidentified by the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a pairwise alignment between (A) ErbB3 and EGFR, and (B)ErbB4 and EGFR used for homology modeling. In the sequence, “*” refersto identical residues; “:” refers to conservative substitutions; and “.”refers to semi-conservative substitutions. The boxes indicatestructurally conserved regions.

FIG. 2 shows stereo views of refined receptor (A) ErbB3 (blue) and (B)ErbB4 (blue) Cα traces superimposed on the x-ray structure of EGFR(red).

FIG. 3 shows ribbon diagrams of (A) NRG-1β/ErbB3 and (B) NRG-1β/ErbB4(B) models. NRG-1β D chain in complex is red. Domains I, II, III, and IVin the receptor are colored blue, green, orange and gray, respectively.Three binding sites in the interface are outlined. The figures wereproduced using the program MOLSCRIPT.

FIG. 4 shows stereo views of (A) the best fit superposition of Cα atomsof the minimized NRG-1β coordinates (blue) on EGF (red) and (B) thealigned amino acid sequences of these proteins.

FIG. 5 shows root mean square deviations for the Cα atoms during thedynamics simulations of complexes of ErbB3 (dash lines) and ErbB4 (solidlines) with NRG-1β.

FIG. 6 shows the interactions between ErbB3 and NRG-1β on the threebinding sites, (A) the interface at site 1; (B) the interface at site 2;(C) the interface at site 3. Only the side chains of interactingresidues are shown. Dotted lines represent hydrogen bonds.

FIG. 7 shows the interactions between ErbB4 and NRG-1β on the threebinding sites, (A) the interface at site 1; (B) the interface at site 2;(C) the interface at site 3. Only the side chains of interactingresidues are shown. Dotted lines represent hydrogen bonds.

FIG. 8 shows (A) the binding energy change for the computational alaninescanning mutagenesis experiments for ErbB3 complex with NRG-1β; and (B)the binding energy change for the computational alanine scanningmutagenesis experiments for ErbB4 complex with NRG-1β. Negative valuesin ΔΔG_(subtotal) indicate highly unfavorable substitutions. Positivevalues in ΔΔG_(subtotal) indicate the preference alanine mutations forthe residues.

FIG. 9 shows phosphorylation of AKT in ErbB2&ErbB4 and ErbB2&ErbB3co-expressing COS7 cells after treatment with neuregulin or its mutation(D43A). In the Figure, Con means the concentration of neuregulin, P-AKTmeans phosphorylation of AKT.

FIG. 10 shows phosphorylation of AKT in ErbB2&ErbB4 and ErbB2&ErbB3co-expressing COS7 cells after treatment with neuregulin or its mutation(L3A). GAPDH is shown as control of protein amount.

FIG. 11 shows phosphorylation of AKT in ErbB2&ErbB4 and ErbB2&ErbB3co-expressing COS7 cells after treatment with neuregulin or its doublemutant (8A47A). GAPDH is shown as control of protein amount.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, “neuregulin” or “NRG” refers to proteins or peptidesthat can bind and activate ErbB2/ErbB4 or ErbB2/ErbB3 heterodimersprotein kinases, such as all neuregulin isoforms, neuregulin EGF domainalone, neuregulin mutants, and any kind of neuregulin-like gene productsthat also activate the above receptors. Neuregulin also includes NRG-1,NRG-2, NRG-3, and NRG-4. These proteins and polypeptides can activatethe above ErbB receptors and modulate their biological reactions, e.g.,stimulate breast cancer cell differentiation and milk protein secretion;induce the differentiation of neural crest cell into Schwann cell;stimulate acetylcholine synthesis in skeletal muscle cell; and improvecardiocyte survival and DNA synthesis. Neuregulin also includes thosevariants with conservative amino acid substitutions that do notsubstantially alter their biological activity. Suitable conservativesubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, TheBejacmin/Cummings Pub. co., p. 224). Neuregulin protein encompasses aneuregulin protein and peptide. Neuregulin nucleic acid encompassesneuregulin nucleic acid and neuregulin oligonucleotide.

As used herein, “epidermal growth factor-like domain” or “EGF-likedomain” refers to a polypeptide motif encoded by the neuregulin genethat binds to and activates ErbB2, ErbB3, ErbB4, or combinationsthereof, and bears a structural similarity to the EGF receptor-bindingdomain as disclosed in WO 00/64400, Holmes et al., Science,256:1205-1210 (1992); U.S. Pat. Nos. 5,530,109 and 5,716,930; Hijazi etal., Int. J. Oncol., 13:1061-1067 (1998); Chang et al., Nature,387:509-512 (1997); Carraway et al., Nature, 387:512-516 (1997);Higashiyama et al., J. Biochem., 122:675-680 (1997); and WO 97/09425.EGF-like domains may be derived from NRG-1, NRG-1, NRG-3, or NRF-4.EGF-like domains may be α or β subtype.

As used herein, a “functional derivative or fragment” of neuregulinrefers to a derivative or fragment of the neuregulin protein or itsencoding nucleic acid that still substantially retains its anti-viralmyocarditis, anti-DCM, anti-cardiotoxic, or anti-myocardial infarctionactivity. Normally, the derivative or fragment retains at least 50% ofits anti-viral myocarditis, anti-DCM, anti-cardiotoxic, oranti-myocardial infarction activity. Preferably, the derivative orfragment retains at least 60%, 70%, 80%, 90%, 95%, 99% and 100% of itsanti-viral myocarditis, anti-DCM, anti-cardiotoxic, or anti-myocardialinfarction activity.

As used herein, “erb” refers to two oncogenes, erb A and erb B,associated with erythroblastosis virus (an acute transformingretrovirus).

As used herein, “an effective amount of a compound for treating aparticular disease” is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. Such amountmay be administered as a single dosage or may be administered accordingto a regimen, whereby it is effective. The amount may cure the diseasebut, typically, is administered in order to ameliorate the symptoms ofthe disease. Repeated administration may be required to achieve thedesired amelioration of symptoms.

As used herein, “treatment” or “treating” refer to any manner in whichthe symptoms of a condition, disorder or disease are ameliorated orotherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein.

As used herein, “amelioration” of the symptoms of a particular disorderby administration of a particular pharmaceutical composition refers toany lessening, whether permanent or temporary, lasting or transient thatcan be attributed to or associated with administration of thecomposition.

As used herein, “production by recombinant means” refers to productionmethods that use recombinant nucleic acid methods that rely on wellknown methods of molecular biology for expressing proteins encoded bycloned nucleic acids.

As used herein, “complementary” when referring to two nucleic acidmolecules, means that the two sequences of nucleotides are capable ofhybridizing, preferably with less than 25%, more preferably with lessthan 15%, even more preferably with less than 5%, most preferably withno mismatches between opposed nucleotides. Preferably the two moleculeswill hybridize under conditions of high stringency.

As used herein: “stringency of hybridization” in determining percentagemismatch is as follows:

1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.;

2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C. (also referred to asmoderate stringency); and

3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.

It is understood that equivalent stringencies may be achieved usingalternative buffers, salts and temperatures.

As used herein, “vector (or plasmid)” refers to discrete elements thatare used to introduce heterologous DNA into cells for either expressionor replication thereof. Selection and use of such vehicles are wellknown within the skill of the artisan. An expression vector includesvectors capable of expressing DNA's that are operatively linked withregulatory sequences, such as promoter regions, that are capable ofeffecting expression of such DNA fragments. Thus, an expression vectorrefers to a recombinant DNA or RNA construct, such as a plasmid, aphage, recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, “a promoter region or promoter element” refers to asegment of DNA or RNA that controls transcription of the DNA or RNA towhich it is operatively linked. The promoter region includes specificsequences that are sufficient for RNA polymerase recognition, bindingand transcription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences that modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be cis actingor may be responsive to trans acting factors. Promoters, depending uponthe nature of the regulation, may be constitutive or regulated.Exemplary promoters contemplated for use in prokaryotes include thebacteriophage T7 and T3 promoters, and the like.

As used herein, “operatively linked or operationally associated” refersto the functional relationship of DNA with regulatory and effectorsequences of nucleotides, such as promoters, enhancers, transcriptionaland translational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itmay be necessary to remove, add or alter 5′ untranslated portions of theclones to eliminate extra, potential inappropriate alternativetranslation initiation (i.e., start) codons or other sequences that mayinterfere with or reduce expression, either at the level oftranscription or translation. Alternatively, consensus ribosome bindingsites (see, e.g., Kozak, J. Biol. Chem., 266:19867-19870 (1991)) can beinserted immediately 5′ of the start codon and may enhance expression.The desirability of (or need for) such modification may be empiricallydetermined.

As used herein, “myocardial infarction” refers to a blockade of acoronary artery or blood flow interruption leading to focal necrosis ofpart of the myocardium caused by severe and persistent ischemia.

B. NRG-1β Variants and Pharmaceutical Compositions

The present invention provides polypeptide variants of NRG-1polynucleotide encoding the polypeptide variants and pharmaceuticalcompositions.

A functional human NRG-1 fragment has the amino acid sequence:

Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asa Gly GlyGlu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys CysPro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe TyrLys Ala Glu Glu Leu Tyr Gln (SEQ ID NO:1) which corresponds to aminoacids 177-237 of human NRG-1.

The human nucleic acid sequence encoding the fragment is

(SEQ ID NO: 2) agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatggaggggagtgc ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgcccaaatgagttt actggtgatc gctgccaaaa ctacgtaatg gcgagcttct acaaggcggaggagctgtac cag

The present invention provides for neuregulin1β variants that compriseamino acid sequence of SEQ ID NO:1 and comprise a different amino acidthan that in SEQ ID NO:1 at residue 3, 8, 16, 25, 29, 31, 33, 35, 43,46, or 47. In certain embodiments, neuregulin1β variants of the presentinvention contain a single amino acid substitute at residue 3, 8, 16,25, 29, 31, 33, 35, 43, 46, or 47 of SEQ ID NO:1. In certainembodiments, neuregulin1β variants of the present invention containmultiple amino acid substitutions at residue 3, 8, 16, 25, 29, 31, 33,35, 43, 46, or 47 of SEQ ID NO:1. In some embodiments, neuregulin1βvariants of the present invention contain two, three, four, five or sixamino acid substitutions at residue 3, 8, 16, 25, 29, 31, 33, 35, 43,46, or 47 of SEQ ID NO:1.

In one aspect, the present invention is directed to a polypeptidevariant of neuregulin-1β comprising amino acid sequence shown in SEQ IDNO:1, wherein the polypeptide variant comprises a different amino acidthan that in SEQ ID NO:1, wherein the polypeptide variant has anenhanced binding affinity to ErbB3 compared to polypeptide of SEQ IDNO:1, and wherein at residue 25 said different amino acid is A, C, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; at residue 35 saiddifferent amino acid is A, C, D, E, F, G, H, I, L, N, M, P, Q, R, S, T,V, W, or Y; and/or at residue 46 said different amino acid is A, C, D,E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y.

In some embodiments, the polypeptide variant consists of the amino acidsequence shown in SEQ ID NO:1, and wherein at residue 25 said differentamino acid is A, C, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, V, Wor Y; at residue 35 said different amino acid is A, C, D, E, F, G, H, I,L, N, M, P, Q, R, S, T, V, W, or Y; and/or at residue 46 said differentamino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, orY.

In some embodiments of the polypeptide variants, at residue 25 saiddifferent amino acid is A; at residue 35 said different amino acid is A;and/or at residue 46 said different amino acid is A.

In some embodiments, the polypeptide variant has a decreased or similaraffinity to an ErbB4 compared to the polypeptide of SEQ ID NO:1. In someembodiments of the polypeptide variants, at residue 25 said differentamino acid is A. In some embodiments of the polypeptide variants, atresidue 35 said different amino acid is A. In some embodiments of thepolypeptide variants, at residue 46 said different amino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βconsisting of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has an enhanced binding affinityto ErbB3 compared to polypeptide consisting of amino acid residues 1-52of SEQ ID NO:1, and wherein at residue 25 said different amino acid isA, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; at residue35 said different amino acid is A, C, D, E, F, G, H, I, L, N, M, P, Q,R, S, T, V, W, or Y; and/or at residue 46 said different amino acid isA, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y.

In some embodiments of the polypeptide variants, at residue 25 saiddifferent amino acid is A; at residue 35 said different amino acid is A;and/or at residue 46 said different amino acid is A.

In some embodiments, the polypeptide variant has a decreased or similarbinding affinity to an ErbB4 compared to the polypeptide consisting ofamino acid residues 1-52 of SEQ ID NO:1. In some embodiments of thepolypeptide variants, at residue 25 said different amino acid is A. Insome embodiments of the polypeptide variants, at residue 35 saiddifferent amino acid is A. In some embodiments of the polypeptidevariants, at residue 46 said different amino acid is A.

In another aspect, the present invention provides a polypeptide variantof neuregulin-1β comprising amino acid sequence shown in SEQ ID NO:1,wherein the polypeptide variant comprises a different amino acid thanthat in SEQ ID NO:1, wherein the polypeptide variant has a decreasedbinding affinity to ErbB4 compared to polypeptide of SEQ ID NO:1, andwherein at residue 3 said different amino acid is A, C, D, E, F, G, H,I, K, M, N, P, Q, R, S, T, V, W, or Y.

In some embodiments, the polypeptide variant consists of the amino acidsequence shown in SEQ ID NO:1, and wherein at residue 3 said differentamino acid is A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY.

In some embodiments of the polypeptide variants, at residue 3 saiddifferent amino acid is A.

In some embodiments, the polypeptide variant has an increased or similarbinding affinity to ErbB3 compared to the polypeptide of SEQ ID NO:1. Insome embodiments of the polypeptide variants, at residue 3 saiddifferent amino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βcomprised of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has a decreased binding affinityto ErbB4 compared to polypeptide consisting of amino acid residues 1-52of SEQ ID NO:1, and wherein at residue 3 said different amino acid is A,C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y.

In some embodiments of the polypeptide variants, at residue 3 saiddifferent amino acid is A.

In some embodiments, the polypeptide variant has an enhanced or similarbinding affinity to an ErbB3 compared to the polypeptide consisting ofamino acid residues 1-52 of SEQ ID NO:1. In some embodiments of thepolypeptide variants, at residue 3 said different amino acid is A.

The invention also provides a polynucleotide comprising a nucleic acidsequence encoding the polypeptide variant described herein that has adecreased binding affinity to ErbB4 compared to polypeptide of SEQ IDNO:1 or polypeptide consisting of amino acid residues 1-52 of SEQ INNO:1.

The invention also provides a pharmaceutical composition comprising aneffective amount of the polypeptide variant described herein that has adecreased binding affinity to ErbB4 compared to polypeptide of SEQ IDNO:1 or polypeptide consisting of amino acid residues 1-52 of SEQ INNO:1, or the polynucleotide encoding the polypeptide variant and apharmaceutically acceptable excipient.

The invention also provides a kit comprising the pharmaceuticalcomposition. In some embodiments, the kit further comprises aninstruction for using the pharmaceutical composition in preventing,treating, or delaying a disease in an individual via activatingErbB2/ErbB3.

The invention also provides a polypeptide variant of neuregulin-1βcomprising amino acid sequence shown in SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has an enhanced binding affinityto ErbB4 compared to polypeptide of SEQ ID NO:1, and wherein at residue16 said different amino acid is A, C, D, E, F, G, H, I, K, L, M, P, Q,R, S, T, V, W, or Y; at residue 29 said different amino acid is A, C, D,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; at residue 31 saiddifferent amino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T,V, W, or Y; at residue 43 said different amino acid is A, C, E, F, G, H,I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and/or at residue 47 saiddifferent amino acid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,V, W, or Y.

In some embodiments, the polypeptide variant consists of the amino acidsequence shown in SEQ ID NO:1, and wherein at residue 16 said differentamino acid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; at residue 29 said different amino acid is A, C, D, E, F, G, H, I, K,L, M, N, Q, R, S, T, V, W, or Y; at residue 31 said different amino acidis A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; atresidue 43 said different amino acid is A, C, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; and/or at residue 47 said different aminoacid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y.

In some embodiments of the polypeptide variants, at residue 16 saiddifferent amino acid is A; at residue 29 said different amino acid is A;at residue 31 said different ammo acid is A; at residue 43 saiddifferent amino acid is A; or at residue 47 said different amino acid isA.

In some embodiments, the polypeptide variant has a decreased or similarbinding affinity to an ErbB3 compared to the polypeptide of SEQ ID NO:1.In some embodiments of the polypeptide variants, at residue 31 saiddifferent amino acid is A. In some embodiments of the polypeptidevariants, at residue 43 said different amino acid is A. In someembodiments of the polypeptide variants, at residue 47 said differentamino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βconsisting of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has an enhanced binding affinityto ErbB4 compared to polypeptide consisting of amino acid residues 1-52of SEQ ID NO:1 and wherein at residue 16 said different amino acid is A,C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; at residue 29said different amino acid is A, C, D, E, F, G, H, I, K, L, M, N, Q, R,S, T, V, W, or Y; at residue 31 said different amino acid is A, C, D, E,F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; at residue 43 saiddifferent amino acid is A, C, E, F, G, H, I, K, L, M, N, P, R, Q, S, T,V, W, or Y; and/or at residue 47 said different amino acid is A, C, D,E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y.

In some embodiments of the polypeptide variants, at residue 16 saiddifferent amino acid is A; at residue 29 said different amino acid is A;at residue 31 said different amino acid is A; at residue 43 saiddifferent amino acid is A; or at residue 47 said different amino acid isA.

In some embodiments, the polypeptide variant has a decreased or similarbinding affinity to an ErbB3 compared to the polypeptide of SEQ ID NO:1.In some embodiments of the polypeptide variants, at residue 31 saiddifferent amino acid is A. In some embodiments of the polypeptidevariants, at residue 43 said different amino acid is A. In someembodiments of the polypeptide variants, at residue 47 said differentamino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βcomprising amino acid sequence of SEQ ID NO:1, wherein the polypeptidevariant comprises a different amino acid than that in SEQ ID NO:1,wherein the polypeptide variant has a decreased binding affinity toErbB3 compared to polypeptide of SEQ ID NO:1 but has a binding affinityto ErbB4 similar to polypeptide of SEQ ID NO:1, and wherein at residue33 said different amino acid is A.

The invention also provides a polypeptide variant of neuregulin-1βconsisting of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has a decreased binding affinityto ErbB3 compared to polypeptide consisting of amino acid residues 1-52of SEQ ID NO:1 but has a binding affinity to ErbB4 similar topolypeptide consisting of amino acid residues 1-52 SEQ ID NO:1, andwherein at residue 33 said different amino acid is A.

The polypeptides of the invention may be produced by chemical synthesisor recombinant methods. Methods of chemically synthesizing polypeptidesare well known in the art. Synthesizing polypeptides using recombinantmethods are also well known in the art and are further described herein.The polypeptides generated may be tested for their binding affinity tothe receptor and activation of the receptor using methods known in theart.

The invention also provides a polynucleotide comprising a nucleic acidsequence encoding any of the polypeptide variants described herein.Polynucleotides complementary to any of the sequences are alsoencompassed by the present invention. Polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials. The polynucleotides of this invention can be obtainedusing chemical synthesis, recombinant methods, or PCR.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Polynucleotidesthat vary due to differences in codon usage are specificallycontemplated by the present invention.

The polynucleotides described herein may be cloned into vectors (such asexpression vectors) and transfected into host cells for production ofthe polypeptides. While the cloning vector selected may vary accordingto the host cell intended to be used, useful cloning vectors willgenerally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, R4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication: one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also provides host cells comprising any of thepolynucleotides described herein. Examples of mammalian host cellsinclude, but are not limited to, COS, HeLa, and CHO cells. See also PCTPublication No. WO 87/04462. Suitable non-mammalian host cells includeprokaryotes (such as E. coli or B. subtillis) and yeast (such as S.cerevisae, S. pombe; or K. lactis).

The invention also provides pharmaceutical compositions comprising anyof the polypeptide variants of NRG-1 or polynucleotides encoding thepolypeptide variants described herein and a pharmaceutically acceptableexcipient or carrier. As used herein, “pharmaceutically acceptableexcipient or carrier” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline or normal (0.9%) saline. Compositions comprising such carriersare formulated by well known conventional methods (see, for example,Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., MackPublishing Co., Easton, Pa., 1990; and Remington, The Science andPractice of Pharmacy 20th Ed. Mack Publishing, 2000). Typically, anappropriate amount of a pharmaceutically-acceptable salt is used in theformulation to render the formulation isotonic. Examples of the carrierinclude saline, Ringer's solution and dextrose solution. The pH of thesolution is preferably from about 5 to about 8, and more preferably fromabout 7 to about 7.5. Further carriers include sustained releasepreparations such as semipermeable matrices of solid hydrophobicpolymers containing the antibody, which matrices are in the form ofshaped articles, e.g., films. liposomes or microparticles. It will beapparent to those persons skilled in the art that certain carriers maybe more preferable depending upon, for instance, the route ofadministration and concentration of the polypeptide and thepolynucleotide being administered.

C. Methods of Screening Polypeptide Variants of NRG-1β

The present invention also provides methods of screening polypeptidevariants of NRG-1. For example, solvent-equilibrated models of theNRG-1β/ErbB3 and NRG-1β/ErbB4 complexes can be constructed using anEGF/EGFR co-crystal structure (see Ogiso, H., et al., Crystal structureof the complex of human epidermal growth factor and receptorextracellular domains. Cell 110, 775-787 (2002), the contents of whichare incorporated by reference), as a starting point, due to the highhomology of this ligand and receptor family. Detailed analysis of theatomic interactions between the interfaces of NRG-1β/ErbB 3 andNRG-1β/ErbB4 is then conducted. The MM-PBSA method is used to calculatethe free energies for the binding of ErbB3 and ErbB4 to NRG-1β. Inaddition, computational alanine-scanning mutagenesis of selectedresidues in the binding-site is performed to rationalize the affinitiesof the two receptors to NRG-1β. The created computational models ofNRG-1β/ErbB3 and NRG-1β/ErbB4 should enable design of NRG-1β variantswith enhanced affinity and selectivity for ErbB4, thus improving theirtherapeutic properties.

The invention provides a method for screening a polypeptide variant ofneuregulin-1β having enhanced binding affinity to ErbB3, which methodcomprises: (a) establishing a three-dimensional structure of aneuregulin-1β or a fragment thereof, an ErbB3, and a complex of theneuregulin-1β or the fragment thereof and the ErbB3 by homologymodeling; (b) establishing data of conformational changes and stabilityof the complex of the neuregulin-1β or the fragment thereof and theErbB3 in solution by molecular dynamics simulation method; (c)calculating subtotal binding free energy (ΔG_(subtotal wildtype)) of theneuregulin-1β or the fragment thereof with the ErbB3 by MolecularMechanics Poisson Boltzmann Surface Area (MM-PBSA) method; (d)calculating subtotal binding free energy(ΔG_(subtotal alanine substituted variant)) of an alanine substitutedvariant of the neuregulin-1β or the fragment thereof with the ErbB3 byMolecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method,wherein the alanine substituted variant comprises an amino acid of theneuregulin-1β or the fragment thereof substituted by an alanine; (e)calculatingΔΔG_(subtotal)=ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);and (f) selecting alanine substituted variant that has a positive valueof ΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3; whereby a polypeptide variant of neuregulin-1βthat has enhanced binding affinity to ErbB3 is identified.

The invention also provides a method for screening a polypeptide variantof neuregulin-1β having enhanced binding affinity selective to ErbB3,which method comprises: (a) establishing a three-dimensional structureof a neuregulin-1β or a fragment thereof, an ErbB3, an ErbB4, a complexof the neuregulin-1β or the fragment thereof and the ErbB3, and acomplex of the neuregulin-1β or the fragment thereof and the ErbB4 byhomology modeling; (b) establishing data of conformational changes andstability of the complex of the neuregulin-1β or the fragment thereofand the ErbB3, and the complex of the neuregulin-1β or the fragmentthereof and the ErbB4 in solution by molecular dynamics simulationmethod; (c) calculating subtotal binding free energy(ΔG_(subtotal wildtype)) of the neuregulin-1β or the fragment thereofwith the ErbB3 or the ErbB4 by Molecular Mechanics Poisson BoltzmannSurface Area (MM-PBSA) method; (d) calculating subtotal binding freeenergy (ΔG_(subtotal alanine substituted variant)) of an alaninesubstituted variant of the neuregulin-1β or the fragment thereof withthe ErbB3 or the ErbB4 by Molecular Mechanics Poisson Boltzmann SurfaceArea (MM-PBSA) method, wherein the alanine substituted variant comprisesan amino acid of the neuregulin-1β or the fragment thereof substitutedby an alanine; (e) calculatingΔΔG_(subtotal)=ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);(f) selecting alanine substituted variant that has a positive value ofΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3, and has a negative value or a value of about zerofor ΔΔG_(subtotal) for the complex of the neuregulin-1β p or thefragment thereof and the ErbB4; whereby a polypeptide variant ofneuregulin-1β that has enhanced binding affinity selective to ErbB3 isidentified. In some embodiments, alanine substituted variant having thenegative value for ΔΔG_(subtotal) for the complex of the neuregulin-1β por the fragment thereof and the ErbB4 is selected, whereby a polypeptidevariant of neuregulin-1β that has enhanced binding affinity to ErbB3 butdecreased binding affinity to ErbB4 is identified. In some embodiments,an alanine substituted variant having the value of about zero forΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB4 is selected, whereby a polypeptide variant ofneuregulin-1β that has enhanced binding affinity to ErbB3 but unchangedbinding affinity to ErbB4 is identified.

In another aspect, the invention provides a method for screening apolypeptide variant of neuregulin-1β having enhanced binding affinity toErbB4, which method comprises: (a) establishing a three-dimensionalstructure of a neuregulin-1β or a fragment thereof, an ErbB4, and acomplex of the neuregulin-1β or the fragment thereof and the ErbB4 byhomology modeling; (b) establishing data of conformational changes andstability of the complex of the neuregulin-1β or the fragment thereofand the ErbB4 in solution by molecular dynamics simulation method; (c)calculating subtotal binding free energy (ΔG_(subtotal wildtype)) of theneuregulin-1β or the fragment thereof with the ErbB4 by MolecularMechanics Poisson Boltzmann Surface Area (MM-PBSA) method; (d)calculating subtotal binding free energy(ΔG_(subtotal alanine substituted variant)) of an alanine substitutedvariant of the neuregulin-1β or the fragment thereof with the ErbB4 byMolecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method,wherein the alanine substituted variant comprises an amino acid of theneuregulin-1β or the fragment thereof substituted by an alanine; (e)calculatingΔΔG_(subtotal)=ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);and (f) selecting alanine substituted variant that has a positive valueof ΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB4; whereby a polypeptide variant of neuregulin-1βthat has enhanced binding affinity to ErbB4 is identified.

The invention also provides a method for screening a polypeptide variantof neuregulin-1β having enhanced binding affinity selective to ErbB4,which method comprises: (a) establishing a three-dimensional structureof a neuregulin-1β or a fragment thereof, an ErbB3, an ErbB4, a complexof the neuregulin-1β or the fragment thereof and the ErbB3, and acomplex of the neuregulin-1β or the fragment thereof and the ErbB4 byhomology modeling; (b) establishing data of conformational changes andstability of the complex of the neuregulin-1β or the fragment thereofand the ErbB3, and the complex of the neuregulin-1β or the fragmentthereof and the ErbB4 in solution by molecular dynamics simulationmethod; (c) calculating subtotal binding free energy(ΔG_(subtotal wildtype)) of the neuregulin-1β or the fragment thereofwith the ErbB3 or the ErbB4 by Molecular Mechanics Poisson BoltzmannSurface Area (MM-PBSA) method; (d) calculating subtotal binding freeenergy (ΔG_(subtotal alanine substituted variant)) of an alaninesubstituted variant of the neuregulin-1β or the fragment thereof withthe ErbB3 or the ErbB4 by Molecular Mechanics Poisson Boltzmann SurfaceArea (MM-PBSA) method, wherein the alanine substituted variant comprisesan amino acid of the neuregulin-1β or the fragment thereof substitutedby an alanine; (e) calculatingΔΔG_(subtotal)=ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);(f) selecting alanine substituted variant that has a positive value ofΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB4, and has a negative value or a value of about zerofor ΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3; whereby a polypeptide variant of neuregulin-1βthat has enhanced binding affinity selective to ErbB4 is identified. Insome embodiments, an alanine substituted variant having a negative valuefor ΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3 is selected, whereby a polypeptide variant ofneuregulin-1β p that has enhanced binding affinity to ErbB4 butdecreased binding affinity to ErbB3 is identified. In some embodiments,an alanine substituted variant having the value of about zero forΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3 is selected, whereby a polypeptide variant ofneuregulin-1β that has enhanced binding affinity to ErbB4 but unchangedbinding affinity to ErbB3 is identified.

The invention also provides a method for screening a polypeptide variantof neuregulin-1β having unchanged binding affinity to ErbB4 but hasdecreased binding affinity to ErbB3, which method comprises: (a)establishing a three-dimensional structure of a neuregulin-1β or afragment thereof, an ErbB3, an ErbB4, a complex of the neuregulin-1β orthe fragment thereof and the ErbB3, and a complex of the neuregulin-1βor the fragment thereof and the ErbB4 by homology modeling; (b)establishing data of conformational changes and stability of the complexof the neuregulin-1β or the fragment thereof and the ErbB3, and thecomplex of the neuregulin-1β or the fragment thereof and the ErbB4 insolution by molecular dynamics simulation method; (c) calculatingsubtotal binding free energy (ΔG_(subtotal wildtype)) of theneuregulin-1β or the fragment thereof with the ErbB3 or the ErbB4 byMolecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method; (d)calculating subtotal binding free energy(ΔG_(subtotal alanine substituted variant)) of an alanine substitutedvariant of the neuregulin-1β or the fragment thereof with the ErbB3 orthe ErbB4 by Molecular Mechanics Poisson Boltzmann Surface Area(MM-PBSA) method, wherein the alanine substituted variant comprises anamino acid of the neuregulin-1β or the fragment thereof substituted byan alanine; (e) calculatingΔΔG_(subtotal)=ΔG_(subtotal wildtype)−ΔG_(subtotal alanine substituted variant);(f) selecting an alanine substituted variant that has a value of aboutzero for ΔΔG_(subtotal) for the complex of the neuregulin-1β or thefragment thereof and the ErbB4 and has a negative value forΔΔG_(subtotal) for the complex of the neuregulin-1β or the fragmentthereof and the ErbB3; whereby a polypeptide variant of neuregulin-1βthat unchanged binding affinity to ErbB4 but has decreased bindingaffinity to ErbB3 is identified.

The invention also provides a polypeptide variant of a neuregulin-1βidentified by the methods described herein.

D. Methods of Using NRG-1β Variants

The invention also provides methods for preventing, treating, ordelaying development of diseases in an individual comprisingadministering to an individual a pharmaceutical composition comprising apolypeptide variant of NRG-1β described herein via activatingErbB2/ErbB3 and/or ErbB2/ErbB4 receptors.

The diseases that can be prevented, treated, or delayed for developmentinclude diseases occurring in bone, ear, eye, eye lid, head, neck,heart, throat, lower jaw, mandibular condyle, upper jaw, mouth, nose,nasal pharynx, oral cavity, pancreas, parotid gland, pinna, pituitarygland, prostate, retina, salivary gland, skin, muscle, bone marrow,thyroid gland, tonsil, neuronal system, respiratory system, digestivesystem, circulatory system, reproductive system, urinary system,endocrine system, cardiovascular system and the hemopoietic system.

As used herein, an “individual” is a mammal, more preferably a human.Mammals include, but are not limited to, farm animals (such as cows,pigs, sheep, goats), sport animals, pets (such as cats, dogs, horses),primates, mice and rats.

In some embodiments, the polypeptide variants of neuregulin-1β used forpreventing, treating or delaying development of a disease has anenhanced binding affinity to ErbB2/ErbB3 receptors. In some embodiments,the polypeptide variants of neuregulin-1β comprise the amino acidsequence shown in SEQ ID NO:1, wherein the polypeptide variant comprisesa different amino acid than that in SEQ ID NO:1, wherein the polypeptidevariant has an enhanced binding affinity to ErbB 3 compared topolypeptide of SEQ ID NO:1, and wherein at residue 25 said differentamino acid is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; at residue 35 said different amino acid is A, C, D, E, F, G, H, I, L,N, M, P, Q, R, S, T, V, W, or Y; and/or at residue 46 said differentamino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, orY. In some embodiments, the polypeptide variant of neuregulin-1βconsists of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has an enhanced binding affinityto ErbB3 compared to polypeptide consisting of amino acid residues 1-52of SEQ ID NO:1, and wherein at residue 25 said different amino acid isA, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; at residue35 said different amino acid is A, C, D, E, F, G, H, I, L, N, M, P, Q,R, S, T, V, W, or Y; or at residue 46 said different amino acid is A, C,D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y. In someembodiments of the polypeptide variants, at residue 25 said differentamino acid is A; at residue 35 said different amino acid is A; and/or atresidue 46 said different amino acid is A.

The diseases that can be prevented, treated, or delayed for developmentvia preferentially activating ErbB2/ErbB3 receptors include nervoussystem diseases (such as central nervous system, peripheral nervoussystem), schizophrenia, bone marrow diseases, menix diseases,demyelinate diseases, extrapyramidal system diseases. In someembodiments, the nervous system disease is schizophrenia.

In some embodiments, the polypeptide variants of neuregulin-1β used forpreventing, treating or delaying development of a disease has anenhanced binding affinity to ErbB2/ErbB4 receptors. In some embodiments,the polypeptide variant of neuregulin-1β comprises the amino acidsequence shown in SEQ ID NO:1, wherein the polypeptide variant comprisesa different amino acid than that in SEQ ID NO:1, wherein the polypeptidevariant has an enhanced binding affinity to ErbB4 compared topolypeptide of SEQ ID NO:1, and wherein at residue 16 said differentamino acid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; at residue 29 said different amino acid is A, C, D, E, F, G, H, I, K,L, M, N, Q, R, S, T, V, W, or Y; at residue 31 said different amino acidis A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; atresidue 43 said different amino acid is A, C, B, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; and/or at residue 47 said different aminoacid is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y. Insome embodiments, the polypeptide variant of neuregulin-1β consists ofamino acid residues 1-52 of SEQ ID NO:1, wherein the polypeptide variantcomprises a different amino acid than that in SEQ ID NO:1, wherein thepolypeptide variant has an enhanced binding affinity to ErbB4 comparedto a polypeptide consisting of amino acid residues 1-52 of SEQ ID NO:1,and wherein at residue 16 said different amino acid is A, C, D, E, F, G,H, I, K, L, M, P, Q, R, S, T, V, W, or Y; at residue 29 said differentamino acid is A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; at residue 31 said different amino acid is A, C, D, E, F, G, H, I, K,L, M, N, P, Q, S, T, V, W, or Y; at residue 43 said different amino acidis A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and/or atresidue 47 said different amino acid is A, C, D, E, F, G, H, I, K, L, M,P, Q, R, S, T, V, W, or Y. In some embodiments of the polypeptidevariant, at residue 16 said different amino acid is A; at residue 29said different amino acid is A; at residue 31 said different amino acidis A; or at residue 47 said different amino acid is A. In someembodiments, the polypeptide variants of neuregulin-1β used forpreventing, treating or delaying development of a disease has a bindingaffinity to ErbB2/ErbB4 receptors similar to the polypeptide of SEQ IDNO:1 or the polypeptide consisting of amino acid residues 1-52 of SEQ IDNO:1, but has decreased binding affinity to ErbB3 than the polypeptideof SEQ ID NO:1 or the polypeptide consisting of amino acid residues 1-52of SEQ ID NO:1. In some embodiments, the polypeptide variant comprisesthe amino acid sequence shown in SEQ ID NO:1, wherein the polypeptidevariant comprises a different amino acid than that in SEQ ID NO:1,wherein the polypeptide variant has a decreased binding affinity toErbB3 compared to the polypeptide of SEQ ID NO:1 but has a bindingaffinity to ErbB4 similar to the polypeptide of SEQ ID NO:1, and whereinat residue 33 said different amino acid is A.

In some embodiments, the polypeptide variants of neuregulin-1β used forpreventing, treating or delaying development of a disease has adecreased binding affinity to ErbB2/ErbB4 receptors. In someembodiments, the polypeptide variant of neuregulin-1β comprises theamino acid sequence shown in SEQ ID NO:1, wherein the polypeptidevariants has a decreased binding affinity to ErbB4 compared topolypeptide of SEQ ID NO:1, and wherein at residue 3 said differentamino acid is A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY. In some embodiments, the polypeptide variant of neuregulin-1βconsists of amino acid residues 1-52 of SEQ ID NO:1, wherein thepolypeptide variant comprises a different amino acid than that in SEQ IDNO:1, wherein the polypeptide variant has an enhanced binding affinityto ErbB4 compared to a polypeptide consisting of amino acid residues1-52 of SEQ ID NO:1, and wherein at residue 3 said different amino acidis A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y. In someembodiments of the polypeptide variant, at residue 16 said differentamino acid is A;

The diseases that can be prevented, treated, or delayed for developmentvia preferentially activating ErbB2/ErbB4 receptors include, but are notlimited to, heart failure, myocardial infarction, dialatedcardiomyopathy, and myocarditis (e.g., viral myocarditis), cardiactoxicity.

The formulation, dosage and route of administration of a polypeptidevariant of neuregulin-1β described herein, or a nucleic acid encodingthe polypeptide variant of neuregulin-1β preferably in the form ofpharmaceutical compositions, can be determined according to the methodsknown in the art (see e.g., Remington: The Science and Practice ofPharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April1997; Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems, Banga, 1999; and Pharmaceutical FormulationDevelopment of Peptides and Proteins, Hovgaard and Frkjr (Ed.), Taylor &Francis, Inc., 2000; Medical Applications of Liposomes, Lasic andPapahadjopoulos (Ed.), Elsevier Science, 1998; Textbook of Gene Therapy,Jain, Hogrefe & Huber Publishers, 1998; Adenoviruses: Basic Biology toGene Therapy, Vol. 15, Seth, Landes Bioscience, 1999; BiopharmaceuticalDrug Design and Development, Wu-Pong and Rojanasakul (Ed.), HumanaPress, 1999; Therapeutic Angiogenesis: From Basic Science to the Clinic,Vol. 28, Dole et al. (Ed.), Springer-Verlag New York, 1999). Apolypeptide variant of neuregulin-1β described herein, or a nucleic acidencoding a polypeptide variant, can be formulated for oral, rectal,topical, inhalational, buccal (e.g., sublingual), parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), transdermaladministration or any other suitable route of administration. The mostsuitable route in any given case will depend on the nature and severityof the condition being treated and on the nature of the particularpolypeptide variant of NRG-1β, or a nucleic acid encoding thepolypeptide variant, which is being used.

A polypeptide variant of neuregulin-1β as described herein, or a nucleicacid encoding a polypeptide variant, can be administered alone.Alternatively and preferably, the polypeptide variant of neuregulin-1β,or a nucleic acid encoding the polypeptide variant, is co-administeredwith a pharmaceutically acceptable carrier or excipient. Any suitablepharmaceutically acceptable carrier or excipient can be used in thepresent method (See e.g., Remington: The Science and Practice ofPharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April1997).

The nucleic acid encoding a polypeptide variant of NRG-1β can be used inthe form of naked DNA, complexed DNA, cDNA, plasmid DNA, RNA or othermixtures thereof as components of the gene delivery system. In anotherembodiment, the nucleic acid encoding a polypeptide variant ofneuregulin-1β, is included in a viral vector. Any viral vectors that aresuitable for gene therapy can be used. For example, an adenovirus vector(U.S. Pat. No. 5,869,305), a simian virus vector (U.S. Pat. No.5,962,274), a conditionally replicating human immunodeficiency viralvector (U.S. Pat. No. 5,888,767), retrovirus, SV40, Herpes simplex viralamplicon vectors and Vaccinia virus vectors can be used. In addition,the genes can be delivered in a non-viral vector system such as aliposome wherein the lipid protects the DNA or other biomaterials fromoxidation during the coagulation.

According to the present invention, a polypeptide variant ofneuregulin-1β described herein, or a nucleic acid encoding a polypeptidevariant, alone or in combination with other agents, carriers orexcipients, may be formulated for any suitable administration route,such as intracavernous injection, subcutaneous injection, intravenousinjection, intramuscular injection, intradermal injection, oral ortopical administration. The method may employ formulations forinjectable administration in unit dosage form, in ampoules or inmultidose containers, with an added preservative. The formulations maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, sterile pyrogen-free water or other solvents, before use.Topical administration in the present invention may employ the use of afoam, gel, cream, ointment, transdermal patch, or paste.

Pharmaceutically acceptable compositions and methods for theiradministration that may be employed for use in this invention include,but are not limited to those described in U.S. Pat. Nos. 5,736,154;6,197,801 B1; 5,741,511; 5,886,039; 5,941,868; 6,258,374 B1; and5,686,102.

The magnitude of a therapeutic dose in the treatment or prevention willvary with the severity of the condition to be treated and the route ofadministration. The dose, and perhaps dose frequency, will also varyaccording to age, body weight, condition and response of the individualpatient.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt or adjust therapy to lower dosage due totoxicity, or adverse effects. Conversely, the physician would also knowhow to and when to adjust treatment to higher levels if the clinicalresponse is not adequate (precluding toxic side effects).

Any suitable route of administration may be used. Dosage forms includetablets, troches, cachet, dispersions, suspensions, solutions, capsules,patches, and the like. See, Remington's Pharmaceutical Sciences.

In practical use, a polypeptide variant of neuregulin-1β, or a nucleicacid encoding a polypeptide variant, alone or in combination with otheragents, may be combined as the active agent in intimate admixture with apharmaceutical carrier or excipient, such as beta-cyclodextrin and2-hydroxy-propyl-beta-cyclodextrin, according to conventionalpharmaceutical compounding techniques. The carrier may take a wide formof preparation desired for administration, topical or parenteral. Inpreparing compositions for parenteral dosage form, such as intravenousinjection or infusion, similar pharmaceutical media may be employed,water, glycols, oils, buffers, sugar, preservatives, liposomes, and thelike known to those of skill in the art. Examples of such parenteralcompositions include, but are not limited to dextrose 5% w/v, normalsaline or other solutions. The total dose of the polypeptide variant ofneuregulin-1β, or a nucleic acid encoding the polypeptide variant, aloneor in combination with other agents to be administered may beadministered in a vial of intravenous fluid, ranging from about 1 ml to2000 ml. The volume of dilution fluid will vary according to the totaldose administered.

The invention also provides for kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically effective amounts of a polypeptide variant ofneuregulin-1β described herein, or a nucleic acid encoding thepolypeptide variant, alone or in combination with other agents, inpharmaceutically acceptable form. Preferred pharmaceutical forms wouldbe in combination with sterile saline, dextrose solution, or bufferedsolution, or other pharmaceutically acceptable sterile fluid.Alternatively, the composition may be lyophilized or dessicated; in thisinstance, the kit optionally further comprises in a container apharmaceutically acceptable solution, preferably sterile, toreconstitute the complex to form a solution for injection purposes.Exemplary pharmaceutically acceptable solutions are saline and dextrosesolution.

In another embodiment, a kit of the invention further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomposition, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of composition by a physician or by thepatient.

E. Examples

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Screening for NRG-1β Variants that Selectively Activate a ErbBReceptor

Methods

1. Construction of 3D Models of the Proteins

The x-ray crystal structure of the human EGF and receptor extracellulardomains (PDB entry 1IVO) and NMR structure of Neuregulin-alpha (NRG-α)epidermal growth factor (EGF)-like domain (PDB entry 1HAF) were obtainedfrom the Protein Data Bank. The amino acid sequences of ErbB3(ERB3_HUMAN), ErbB4 (ERB4_HUMAN), and NRG-1β (NRG1_HUMAN) were obtainedfrom the Swiss_Prot/TrEMBL database.

All models were generated using the Homology module of the Insight IIsoftware (Accelrys Corporate, San Diego, Calif.). The NMR coordinates ofNeuregulin-α were used to model the initial structure of Neuregulin-1β(NRG-1β₁₇₇₋₂₂₉) The pairwise sequence alignments between the targetsequences of ErbB3 and ErbB4 and the template EGFR sequence wereperformed against each of the homology sequences, respectively. Theposition of the disulphide bonds between the cysteine residues wasachieved using the aligned cysteine residues of the target and thecorresponding disulphide bridges in the template. Gaps were insertedinto the sequences to find an optimal alignment with alength-independent gap penalty of 6. The final sequence alignments ofErbB3 and ErbB4 with EGFR are shown in FIG. 1. Regions of the structuralsimilarity were automatically selected and employed to build a frameworkfor the model structure. Wherever the two aligned residues were thesame, the template side-chain geometry was adopted in the target.Otherwise, the side chain in the template was replaced by that ofcorresponding target residue by aligning the C_(α)-C_(β) bonds. Gapregions were generated by searching for a suitable fragment from proteindatabank screening and the in-house database. The final loopconformation was chosen from one of the top 10 structures that had thelowest root mean square (RMS) values with compatible geometries betweenthe target and a suitable fragment. Some missing atoms were also addedto incomplete side chains of each of these models using the Biopolymermodule in Sybyl to ensure proper assignment of hydrogens and atomiccharge. The preliminary models were then subject to evaluation byPROCHECK and Profiles-3D to examine the stereo-chemical quality and theprotein structure of the models. In the model evaluation by Profiles-3D,a self-compatibility score (S) was determined for each residue in thesequence.

After the initial models of ErbB3, ErbB4 and NRG-1β were builtseparately, NRG-1β/ErbB3 and NRG-1β/ErbB4 complexes were constituted bysuperimposing the models with the EGF/EGFR complex. The resultingstructures were optimized by energy minimization using the Amber forcefield implemented in the Sybyl software package. The backbone atoms ofall model residues were first fixed during the initial stages ofrefinement to prevent the backbone from deforming significantly. Thenthe number of constraints applied to the model was reduced duringsubsequent cycles. For minimization the following parameters were used:Kollman-united-atom force field and united-atom charge,distant-dependent dielectric constant of 4.0 R, 9.5 Å cutoff fornonbonded calculations, and conjugate gradient minimization until theroot-mean-square (RMS) gradient in the energy was less than 0.05kcal/mol·Å. The refined models were then subjected to molecular dynamicssimulations.

2. Estimation of Ligand-Binding Contribution by Molecular DynamicsSimulations of NRG-1β in Complex with the Receptor

Several sets of molecular dynamics (MD) simulations were performed onprotein complexes and mutant structures separately using the AMBER 7.0simulation package and the Parm99 force field. The complex structureswere solvated using a box of TIP3P water molecules extending at least 10Å away from the boundary of any protein atoms. The NRG-1β/ErbB3structure was solvated in a 100×96×83 Å box of 7976 TIP3P water, and theNRG-1β/ErbB4 structure was solvated in a 104×102×87 Å box of 8075 TIP3Pwater molecules. An appropriate number of counterions were added toneutralize the system. The Particle Mesh Ewald (PME) method was employedto calculate the long-range electrostatics interactions. All the MD runswere set up using the same protocol. First, the solvent complexes weresubjected to 200 steps of minimization using the steepest descent methodfollowed by conjugate gradient to remove close van der Waals contacts.Then, a second minimization of 500 steps was performed on the entireprotein-ligand-water complexes.

The relaxed structures were then subjected to MD simulations. Eachsystem was gradually heated from 0 to 300 K in 15 ps with threeintervals, and then equilibrated for 25 ps at 300 K, followed by a datacollection run, giving a total simulation time of 1100 ps forNRG-1β/ErbB3 and 900 ps for NRG-1β/ErbB4. The non-bonded cutoff was setto 8.0 Å and the nonbonded pairs were updated every 25 steps. The SHAKEmethod was applied to constrain all covalent bonds involving hydrogenatoms. Each simulation was coupled to a 300K thermal bath at 1.0 atmpressure by applying the algorithm of Berendsen. The temperature andpressure coupling parameters were set as 0.2 ps and 0.05 ps,respectively. An integration time step of the molecular dynamicscalculations was 2 fs. In the energy minimizations and moleculardynamics simulations, periodic boundary conditions were applied in alldirections. MD simulations were run on an SGI Origin3800 computer at theShanghai Institute of Materia Medica.

The analyses of the simulations focused on the production stages. Theroot mean square deviations (RMSDs) of the backbone were calculated fromthe trajectories at 1 ps intervals, with the initial structure as thereference. The root mean square fluctuation-(RMSF) of each residue wascalculated similarly. The binding interactions between receptors andligand were analyzed on the completed models using the program LIGPLOT.

3. Calculation of Free Energy of NRG-1β in Complex with the Receptor byMM-PBSA Method

Coordinates from the dynamic trajectory were used every 4 ps (100snapshots out of 400 ps were processed), and the MM-PBSA calculation wasperformed on each of them using the AMBER 7.0 program. For each snapshotcollected during the simulation, the ligand-protein binding free energy(ΔG_(binding)) was calculated using Eq. (1):ΔG _(binding) =ΔG _(complex) −[ΔG _(protein) +ΔG _(ligand)]  (1)where ΔG_(complex), ΔG_(protein) and ΔG_(ligand) are the free energiesof the complex, protein and ligand. Each free energy term in Eq. (1) wascalculated with the absolute free energy of the species (protein, ligandand their complex) in gas phase (E_(gas)), the solvation free energy(ΔG_(solvation)) and the entropy term (TΔS) using Eq. (2):ΔG=E _(gas) +ΔG _(solvation) −TΔS  (2)E_(gas) is the sum of the internal strain energy (E_(int)), van derWaals energy (E_(vdw)) and electrostatic energy (E_(electrostatic)) (Eq.(3)) E_(int) is the energy associated with vibrations of covalent bondsand bond angles, rotation of single bond torsional angles (Eq. (4)).E _(gas) =E _(int) +E _(vdw) +E _(electrostatic)  (3)E _(int) =E _(bond) +E _(angle) +E _(torsion)  (4)The solvation free energy, ΔG_(solvation), is approximated as the sum ofthe polar contribution (G_(PB)) and nonpolar contribution (G_(nonpolar))using a continuum representation of the solvent:ΔG _(solvation) =G _(PB) +G _(nonpolar)  (5)The polar contribution (G_(PB)) to the solvation energy was calculatedusing the DELPHI program with PARSE atom radii and standard Parm94charges for amino acids. The grid size used was 0.5 Å. The dielectricconstant was set to 1 for interior solute and 80 for exterior water. Atotal of 1000 iterations were performed for each G_(PB) calculation toachieve a better convergence. The nonpolar contributions (G_(nonpolar))were estimated using a simple equation: G_(nonpolar)=γ×SASA+b kcal/mol.SASA is the solvent-accessible surface area that was estimated using theMSMS algorithm with probe radius of 1.4 Å. The surface tensionproportionality constant γ and the free energy of nonpolar salvation fora point solute b were set to 0.00542 kcal mol⁻¹ Å⁻² and 0.092 kcalmol⁻¹, respectively.

The entropy calculation is extremely time-consuming for large systems.In this study, only ΔG_(subtotal) (without term of −TΔS) was estimatedto address the mutation effect to the binding free energy.

4. Analysis of Computational Alanine-Scanning Mutagenesis

The computational alanine-scanning method was applied to estimate therelative binding affinity of different NRG-1β variants to ErbB3 andErbB4, respectively. Two methods were used to calculate the relativebinding strength of different peptide mutants. In the first approach,the structures of complex, protein and peptide were taken from the samesnapshot of the complex trajectories. The alanine mutant structures weregenerated by altering the coordinates of the wild-type trajectory. Thismethod involved deleting atoms and truncating the mutated residue atC_(γ) by replacing with a hydrogen atom. All parameters in the topologyfiles for the mutated residues were accordingly replaced with thealanine residue parameters. A total of 100 snapshots out of 400 ps(every fourth snapshot) were obtained for energy calculation. Therelative binding free energy is the free energy difference between thewild-type and alanine mutated.

In the second method for calculating the relative binding free energy,we conducted several sets of separate dynamics simulations for thealanine mutated NRG-1β peptides in the complexes with ErbB3 and ErbB4,respectively. Then the energy components were calculated from 100snapshots out of 400 ps collected trajectories (every fourth snapshot)and statistical analyses were carried out.

Results

1. Modeling Results and Evaluation

The 3D models of ErbB3 and ErbB4 were constructed by homology modelingbased on the x-ray crystal structure of the complex formed between humanepidermal growth factor (EGF) and the extracellular domain of the EGFreceptor (EGFR). FASTA and BLAST searches of sequence databases producedseveral structures that are homologous to ErbB3 and ErbB34. Within theEGFR family, we found that the x-ray structures of unliganded ErbB3 (PDBentry 1M6B), inactivated EGFR in complex with EGF (PDB entry 1NQ1),ErbB2 bounded by Herceptin Fab (PDB entry 1N8Z) and the complex ofEGF/EGFR (PDB entry 1IVO) were among the homologous proteins with highersequence identities to ErbB3 and ErbB4. ErbB2 is an unusual receptor ofthe EGFR family for which no high-affinity ligand has been found. Theunliganded ErbB3 has an identical sequence compared with the receptor ofNRG-1β/ErbB3 complex. To focus on ligand/receptor interactions, thestructure of the EGF/EGFR complex was selected as the template forstructural modeling.

On the basis of internal sequence conservation and identity among theEGF receptor family members, the extracellular domain has beenclassified into four domains (I to IV). Domains II and IV are richer incysteine residues, containing 24 and 21 cysteines, respectively. In thecrystal structure of EGFR, most of domain IV (residues 513 to 619) isstructurally disordered. Therefore, the region of domain IV was excludedin our homology modeling. The sequences of ErbB3 (residues 8-511) andErbB4 (residues 25-533) have 44% and 48% identity with EGFR (residues3-512). Furthermore, another 40% of these residues were conservativesubstitutions, giving an overall sequence similarity of about 80% forboth ErbB3 and ErbB4 to EGFR as shown in FIG. 1.

The accuracy of a model can vary significantly even within differentregions of the same protein: usually highly-conserved regions can bemodeled much more reliable than the variable loops or surface residues.A total of 7 and 4 structurally conserved regions (SCRs) were deducedfor ErbB3 and ErbB4 respectively, which constituted major segments ofthe total sequences (FIG. 1). Coordinates for the remaining parts of thesequence, designated structurally variable regions (SVRs), weregenerated by searching a protein structure database as described abovein the Methods. FIG. 2 shows stereo plots of the two refined modelssuperimposed with the x-ray structure of EGFR. It is clear that theoverall topology of the template protein has been inherited while somesignificant local conformation changes are presented in the variableregions. The C_(α) backbone root mean square deviations (RMSDs) of therefined models from their template are small, 0.55 Å for ErbB3 and 0.39Å for ErbB4, indicating a high degree of structural similarity, asexpected from their high degree of sequence homology.

Several key residues of this receptor family such as Leu69, Leu98,Val350, Asp355 and Phe357 in EGFR which interact hydrophobically withresidues of EGF are conserved in ErbB3 and ErbB4 as well. EGFR has astack of glycine residues at positions 39, 63, 85, and 122 in domain 1and 343, 379, 404, and 435 in domain III. These residues areconservatively retained in corresponding conformations in the ErbB3 andErbB4 models. Like EGFR, both domains I and III of ErbB3 and ErbB4comprise six turns of a β-helix capped at each end by a helix and adisulfide bond. In domains I and III of ErbB3 and ErbB4, there are alsoconserved tryptophans (Trp176, Trp492 for ErbB3 and Trp198, Trp513 forErbB4) inserted between the fourth and fifth turns of the β-helix.Domain II of the receptors has a similar fold to the second domain ofEGFR, which is composed of several small modules with similardisulphide-bond connectivities.

The models were then evaluated by a Ramachandran plot structurevalidation test using PROCHECK. The Ramachandran plot analyzes thebackbone phi (φ), psi (ψ) torsional angles of a protein and calculatesthe percentage occupancies for “favoured” “allowed”, “generouslyallowed,” and “disallowed” regions as a quality measurement of a proteinstructure. The values obtained for the models of ErbB3 and ErbB4 aresummarized in Table 1 below. Altogether 96.2% and 97.5% of the residueswere in favoured and allowed regions for the models of ErbB3 and ErbB4,respectively. In addition to the evaluation of stereochemical qualityfor the models, we also performed Profiles-3D analyses to furtherevaluate the models by checking the quality of side-chain packing. Theoverall quality scores for ErbB3 and ErbB4 are 230.51/257.48 and216/256.56 respectively, where the denominator denotes the expectedscore for a protein of this length based on known structures. Forcomparison, scores of 115.87/257.48 and 115.45/256.56 or less, wouldsuggest the structure is almost certainly incorrect. After successfulvalidation, the quality of ErbB3 and ErbB4 structures appear to beacceptable for further study.

TABLE 1 Ramachandran plot calculations on 3D models of ErbB3 and ErbB4computed with the PROCHECK program. ErbB3 ErbB4 % of residues in mostfavoured regions 68.0 73.9 % of residues in additional allowed zones28.2 23.6 % of residues in generously allowed regions 2.3 0.8 % ofresidues in disallowed regions 1.5 1.6 % of non-glycine and non-prolineresidues 100.0 100.0

A homology model for NRG-1β(₁₇₇₋₂₂₉) was constructed from the NRG-αstructure (PDB entry 1HAF) with an overall sequence identity of 80%. Forconvenience, we chose to number the residues in this NRG-αdomainsequentially from 1 to 63. The C-terminal residues 51 to 63 aredisordered and flexible in the solution structure of NRG-α. The moreordered region (residues 1-50) has been shown to be the minimal subunitrequired for binding to cellular receptors. Thus, a truncated model ofNRG-1β, consisting of residues 1-52, was built as described in themethod. The model includes an N-terminal subdomain containing a centralthree-stranded β-sheet, a helical region, and a short β-sheet in theC-terminal subdomain. NRG-1β is stabilized by three disulfide bridges,Cys6-Cys20, Cys14-Cys34 and Cys36-Cys45.

2. Modeling of NRG-1β Binding to Receptors

The final 3D models of the NRG-1β/ErbB3 and NRG-1/ErbB4 complexes areshown in FIG. 3. In each complex, domains I, II and III of the receptorsare arranged in a C shape, and NRG-1β is accommodated between domains Iand III in a similar way of EGF binding to EGFR (FIG. 3). This isconsistent with the previous biochemical results that domains I and IIIof ErbB3 may be involved in the NRG-1β binding. The binding affinitystudies suggest that the binding determinants on the ErbB3 and ErbB4receptors are very similar although they have substantial overallsequence diversity. Mutagenesis studies also revealed that both the N-and C-termini of NRG-1β are important for ligand-receptor binding. Thesetwo regions are far away in distance from each other in the foldedprotein, supporting the role of multiple regions of ligand-receptorinteraction. The intervening residues, in particular those that areconserved between NRG-1β and EGF, may play purely structural roles bymaintaining the appropriate distance and orientation between these tworegions. The X-ray structure of the unliganded ErbB3 shows that the sizeof the ligand binding site between domains I and III is twice as big asthe ligand size when domains II and IV interact. The domain II and IVcontact seems to constrain the relative orientations of domains I andIII for ligand binding. Thus, a ligand that contacts only to domain I(or domain III) would bind the receptor but fail to induce the domainarrangement that seems necessary for signaling. Based on these results,we can conclude that there are at least two receptor binding sitesrequired for signal required of transduction.

Comparison of the NRG-1β structure with EGF revealed a high degree ofstructural similarity (FIG. 4). Excluding the N-terminal region(Ser1-Lys5), the disordered Ω-loop (Lys24-Ser30) and C-terminal region(Met50-Ser52), the C_(α) atoms of NRG-1β aligns well with that of EGF,with an RMSD of ˜1.3 Å. NRG-1β has a three-residue insertion relative tothe sequence of EGF. However, the substitution of residues 21-33 in theNRG-1β with residues 21-30 of EGF has no effect on neuregulin receptorbinding or its ability to stimulate receptor phosphorylation. Theexperimental results suggested that the functional significance of thethree-residue (residues 28-30) insertion in die NRG-1β is minimal,despite the significant structural differences in that region.Therefore, the orientation of the extended loop in NRG-1β would notaffect the modeling of NRG-1βbinding to the receptors.

3. Molecular Dynamics Simulations

The behavior of the receptor-ligand complexes was studied by moleculardynamics simulations to account for protein flexibility andconformational changes. The starting models of NRG-1β/ErbB3 andNRG-1β/ErbB4 were subjected to 1.1 ns and 0.9 ns of MD simulations,respectively. The root mean square deviations (RMSDs) of C_(α) atomsfrom their initial positions (t=0 ps) have been used to measure thestability and to gain insight into possible structure fluctuation.

The time evolution of the C_(α) atom RMSDs of NRG-1β/ErbB3 andNRG-1β/ErbB4 complexes is presented in FIG. 5. In the plot, a sharp risewas observed during the first 100 ps in all residue RMSDs and then ittends to flatten out. The magnitude of these RMSD curves, however, didnot continue to increase during the data collection period, implyingthat the structures of NRG-1β/ErbB3 and NRG-1β/ErbB4 were stable overthis time scale. The average RMSDs was below 3.0 Å over the entiresimulation for both NRG-1β/ErbB3 and NRG-1β/ErbB4 complexes.Particularly, the simulation trajectories of the ErbB4 with ligandNRG-1β appeared to be well equilibrated with an average RMSD value of2.6 Å over the last 400 ps. The NRG-1β bound ErbB3 structure showed ahigher RMSD with average value of 3.7 Å during the last 400 pssimulation. This is indicative of the relative stability of the NRG-1βbounded structures. This trend was again apparent in the analysis ofresidue-wise RMS fluctuations. The residues of ErbB3 in the NRG-1β boundstructure fluctuate more about their mean positions than the residues inthe ErbB4 complex.

Superimposition of the average structures of all trajectories with theirrespective starting structures revealed regions of major conformationalchange. Residues that showed the largest deviations in NRG-1β/ErbB3structure (residues 243-256) are the same as that in the NRG-1β/ErbB4complex (residues 265-278), but to a great extent. These residues are apart of β-hairpin loop that extends nearly 20 Å from domain II in thex-ray structure of ErbB3. This β-hairpin loop is highly conserved withinthe EGFR family and plays a dominant role not only in intramolecularcontact between domains II and IV but also in intermolecularreceptor-receptor interaction. Our 1:1 truncated complex models whichlack of interactions between domains II and IV as well asreceptor-receptor contacts may result in considerably high RMSDs at thisregion. But overall analyses show that the modeled structures remainstable along the simulations without suffering remarkable structuralchanges.

4. Ligand-Receptors Interactions

The ligand-receptor interaction on each receptor consists of three sitesas in the interaction in EGF/EGFR, which is designated hereafter as site1 in domain I and sites 2 and 3 in domain III of the receptor (FIG. 3).Residues 20-35 and Leu3 of NRG-1β interact with site 1. The regioncontaining residues 10-19 and Arg44 of NRG-1β interacts with site 2. TheC-terminal region around residue Tyr48 interacts with site 3. Theseresidues of ligand form a variety of electrostatic, hydrophobic, andhydrogen-bonding interactions to the receptor.

From the analysis of hydrogen bond trajectories between the ligand andthe receptor, a few key interactions mediated by hydrogen bonds betweenside chain residues of NRG-1β/ErbB3 and NRG-1β/ErbB4 have been detected.These hydrogen bonds are listed along with distances in Table 2. Fromthis Table 2, it can be seen that the crucial interactions includingthose of residues Arg44 and Tyr48 in NRG-1β(Arg41 and Arg45 in EGF) withthe receptors are retained. The Arg44 side chain of NRG-1β makes ahydrogen bond with the Asp352 side chain of ErbB3 and the Asp376 sidechain of ErbB4, respectively. This result is supported by theexperimental finding that the replacement of Arg44 by alaninesignificantly reduced the ErbB3 and ErbB4 binding activity of NRG-1β.

TABLE 2 Hydrogen bonds between residue side chains of ligand andreceptor. Hydrogen bond partners NRG-1β ErbB3 Residue Group ResidueGroup Distance (Å) Asn47 ND2 Tyr405 OH 2.93 Tyr48 OH Asn379 ND2 3.12Tyr48 OH Asn379 OD1 3.10 Arg44 NH2 Asp352 OD2 2.61 Arg44 NH1 Asp352 OD12.67 Asn47 ND2 Asp343 OD1 2.90 Arg31 NH2 Glu131 OE2 2.75 Arg31 NH1Glu131 OE1 2.92 Asn38 ND2 Asn25 OD1 3.25 NRG-1β ErbB4 Residue GroupResidue Group Distance (Å) Glu39 OE2 Lys438 NZ 2.87 Tyr48 OH Asn403 OD12.73 Arg44 NH2 Asp376 OD1 3.04 Arg44 NH1 Asp376 OD1 3.12 Ser27 OG Asp150OD1 3.59 Asn28 ND2 Tyr148 OH 3.16 Lys35 NZ Ser40 OG 2.82 Asp43 OD2 Lys35NZ 2.81

Besides the hydrogen bonds, the hydrophobic interactions between NRG-1βand the interfaces of sites 1, 2 and 3 of ErbB3 and ErbB4 are extensive.

In NRG-1β/ErbB3 complex, the side chains of Val47, Leu48, Met72, andLeu102 in site 1 of ErbB3 hydrophobically interact with Leu3, Val23 andLeu33 of NRG-1β (FIG. 6A). The Trp354 side chain in site 2 of ErbB3hydrophobically interacts with Phe13 and Tyr32 of NRG-1β (FIG. 6B).Furthermore, the long aliphatic portion of the Arg44 (NRG-1β) side chainalso provides van der Waals contacts with the Trp354 (ErbB3) side chain.The side chains of Tyr405, Phe409 and Ile413 in site 3 of ErbB3 form ahydrophobic interaction network with that of residues around Tyr48 ofNRG-1β (FIG. 6C).

Similar hydrophobic interactions are observed in the NRG-1β/ErbB4complex. As a result of these interactions, three hydrophobicinteraction networks are formed on the interfaces between NRG-1β andErbB4. The side chains of Leu3, Val23 and Leu33 of NRG-1β form van derWaals contacts with Leu36, Leu91 and Leu121 in site 1 of ErbB4 (FIG.7A). The side chains of Phe13, Val15 and residues around Tyr48 of NRG-1βhave similar interactions with sites 2 and 3 of ErbB4 (FIGS. 7B and 7C),respectively. Both putative interactions identified from refined modelsare in agreement with the mutagenesis studies of NRG-1β in the presenceof ErbB3 or ErbB4.

The binding analysis of NRG-1β with receptors presented above providesus with rich information about the mechanism by which ErbB3 and ErbB4interacts with NRG-1β. This in turn facilitates our ability tounderstand and further optimize the interaction components for NRG-1βbinding and stability to the receptor by computational mutagenesisapproach.

5. Computational Alanine-Scanning Mutagenesis of NRG-1β by MM-PBSAMethod

To determine the contribution of each residue in the interactioninterface to the ligand-receptor binding and to identify key residues inthe ligand that could potentially increase its binding affinity uponmutation, computational alanine-scanning mutagenesis was performed onNRG-1β and the binding free energies of the mutated ligands to thereceptors were calculated using the MM-PBSA method. To reducecomputational time, the entropy contribution (TΔS) to the binding freeenergy was not calculated in this study. The entropy contributions areexpected to be canceled when the differences in the binding freeenergies are calculated between the wild type and mutants. Thisassumption seems reasonable, as demonstrated by Massova and Kollman'scalculation of TΔS for a 12-residue peptide derived from human p53 andits mutants. Therefore, only ΔG_(subtotal) (without the −TΔS term) wasused as the criteria for the ΔG_(binding) estimates.

Table 3 shows all the energy terms and the subtotal binding freeenergies of NRG-1β and its mutants to ErbB3 and ErbB4, respectively. Asseen in Table 3, the intermolecular van der Waals interaction and thenonpolar solvation term provide the driving force for the binding. Bothcomplex formations lead to strongly favorable electrostatic interactions(E_(ele)), opposed by unfavorable contributions due to the polar part ofsalvation free energy (G_(PB)). Similar results can be found in severalother studies. The total electrostatic contributions (E_(ele)+G_(PB))for NRG-1β/ErbB3 and NRG-1β/ErbB4 are 110.2 kcal mol⁻¹ and 112.5 kcalmol⁻¹, respectively, and thus disfavor complex formation, again inagreement with related studies cited above.

TABLE 3 Results of free energy calculation of NRG1-β with ErbB receptorsby MM-PBSA^(a) Contribution NRG-1β/ErbB3 ErbB3 NRG-1β Delta^(b) Std^(d)Mean^(c) Std^(d) Mean^(c) Std^(d) Mean^(c) Std^(d) Mean^(c) Std^(d)E_(ele) −16753.91 112.64 −14940.46 102.75 −1361.15 22.58 −452.30 25.80E_(vdw) −2006.94 32.49 −1739.60 31.19 −123.33 10.46 −144.02 7.29 E_(int)10831.80 63.57 9798.05 60.27 1033.75 20.77 0.00 0.00 E_(gas) −7929.04133.98 −6882.00 123.65 −450.73 25.50 −596.31 25.25 G_(nonpolar) 143.011.19 132.17 1.01 19.13 0.18 −8.28 0.44 G_(PB) −7440.22 101.09 −6863.6188.32 −1139.12 18.86 562.52 25.27 G_(sol) −7297.21 100.53 −6731.44 87.93−1120.00 18.84 554.23 25.12 G_(subtotal) −15226.25 66.86 −13613.45 65.36−1570.72 18.19 −42.08 7.21 -TΔS ND ND ND ND ND ND ND ND ContributionNRG-1β/ErbB4 ErbB3 NRG-1β Delta^(b) Std^(d) Mean^(c) Std^(d) MeanStd^(d) Mean^(c) Std^(d) Mean^(c) Std^(d) E_(ele) −16888.05 94.37−15171.74 66.74 −1343.04 33.13 −373.27 27.41 E_(vdw) −2068.19 31.77−1815.76 29.10 −119.74 10.70 −132.69 6.88 E_(int) 10794.92 61.48 9749.4656.82 1045.46 19.44 0.00 0.00 E_(gas) −8161.32 117.81 −7238.04 90.27−417.32 43.40 −505.97 26.96 G_(nonpolar) 138.41 1.45 126.65 1.24 19.440.41 −7.68 0.52 G_(PB) −7327.22 94.47 −6658.67 67.14 −1153.33 36.53485.77 25.23 G_(sol) −7188.82 93.77 −6532.02 66.67 −1134.89 36.22 478.0925.09 G_(subtotal) −15359.14 68.76 −13770.06 63.84 −1552.20 20.79 −27.8810.09 -TΔS ND ND ND ND ND ND ND ND ^(a)All value are given in kcalmol⁻¹. ^(b)Contribution (Complex)-Contribution (Receptor)-Contribution(ligand) ^(c)Average over 400 snapshots. ^(d)Standard error of meanvalues.

Based on molecular dynamics simulations of ligand-receptor interactions,we have identified key residues of NRG-1β responsible for binding toreceptor residues. Table 4 shows the results of the computationalalanine-scanning mutagenesis approach for 20 residues of the total 52residues of NRG-1β which contribute to the ErbB3 and ErbB4 binding. Thepositive and negative values ofΔΔG_(subtotal)(ΔG_(wildtype)−ΔG_(mutant)) indicate favorable andunfavorable substitutions, respectively. Data are also depicted as agraph in FIG. 8. The results in FIG. 8 show that several alanine mutantsof NRG-1β have significantly reduced ligand interaction energies withErbB3 and ErbB4, especially at positions 44, 48 and 50. This can beexplained structurally. Residues Arg44 and Tyr48 form hydrogen bondswith polar residues of the receptors (see FIGS. 6 and 7), which arereflected by the major contribution of electrostatic interactions to thebinding free energy (ΔΔE_(ele) term; see supplementary Tables 1 and 2).Arg44Ala and Tyr48Ala mutations abolish the important hydrogen bondsbetween the ligand and receptors. Tyr48Ala and Met50Ala mutations causesignificant loss of favorable van der Waals interactions between theligand and receptors (ΔΔE_(vdw) term; see supplementary Tables 1 and 2).

TABLE 4 Computational alanine-scanning mutagenesis results for NRG-1βcomplex with ErbB3 and ErbB4 (ΔΔG_(subtotal) = ΔG_(wildtype) −ΔG_(mutant)) NRG-1β/ErbB3 NRG-1β/ErbB4 NRG-1β ΔΔG_(subtotal)ΔΔG_(subtotal) Position (kcal/mol) (kcal/mol) His2Ala −0.10 ± 0.54  0.13± 1.90 Leu3Ala −1.14 ± 1.24 −0.87 ± 1.19 Phe13Ala −1.06 ± 0.76 −0.25 ±0.90 Val15Ala −0.78 ± 0.86 −1.19 ± 0.63 Asn16Ala  0.78 ± 0.85  1.90 ±2.94 Val23Ala −0.50 ± 1.60 −1.53 ± 1.00 Asp25Ala  2.26 ± 0.66 −2.20 ±3.71 Arg31Ala −5.90 ± 1.14  1.83 ± 4.21 Tyr32Ala  0.32 ± 0.57 −0.34 ±0.01 Leu33Ala −3.31 ± 0.38 −0.32 ± 1.27 Lys35Ala  1.13 ± 0.93  0.00 ±2.09 Asn38Ala  0.59 ± 0.76  1.31 ± 0.89 Glu39Ala  0.75 ± 2.09 −0.51 ±2.67 Phe40Ala  1.59 ± 2.12  1.08 ± 3.50 Arg44Ala −5.43 ± 4.79 −5.30 ±2.77 Gln46Ala  3.57 ± 2.69 −1.01 ± 1.62 Asn47Ala −0.07 ± 2.22  2.03 ±1.72 Tyr48Ala −2.99 ± 2.45 −3.05 ± 2.40 Met50Ala −3.45 ± 0.54 −1.92 ±1.45 Ser52Ala −0.21 ± 1.77  0.49 ± 2.25

To gain further insight into the contributions of each alanine mutation,the change of the intermolecular van der Waals and electrostaticinteractions plus the polar solvation free energies upon alaninemutation is shown in FIG. 8. In most cases the change of intermolecularelectrostatic energies is anticorrelated with the change of polarsolvation free energies. Therefore, it is important to combine ΔΔE_(ele)and ΔΔG_(PB) together based on the common electrostatic origin of thecontributions. From FIG. 8, the ΔΔG_(nonpolar) is very small compared tochanges in other free energy components. The contributions of theΔΔE_(vdw) are mostly negative, which indicate that alanine substitutionreduces van der Waals contacts on the binding interfaces. On the otherhand, the terms which are the combined of ΔΔE_(ele) and ΔΔG_(PB) aremost favored by Ala mutations. One exception is the substitution ofArg44, which loses both van der Waals and electrostatic(ΔΔE_(ele)+ΔΔG_(PB)) interactions. The intermolecular interactionsbetween the positively-charged Arg44 of NRG-1β and negatively-chargedAsp352 of ErbB3 and Asp376 of ErbB4 are strongly favored in the wildtype complexes. A similar result is observed for the Arg31Alasubstitution of NRG-1β interacting with ErbB3 (FIG. 8A), but not withErbB4 (FIG. 8B). The structure of NRG-1β/ErbB 3 reveals that the Arg31side-chain forms two hydrogen bonds to the Glu131 side chain of ErbB 3(Table 2). Therefore, Arg31Ala mutant lacks the hydrogen bondinginteraction. However, Arg31 of NRG-1β is not involved in hydrogenbonding to the receptor of ErbB4 (Table 2).

6. Comparative Studies of ΔΔG_(binding) from the Separate Trajectoriesof NRG-1β Arg31Ala and Asn47Ala Mutations

To check the validity of the assumption whether the alanine mutations donot cause large conformational changes for the global structure ofNRG-1β/ErbB3 and NRG-1β/ErbB4 complexes, two molecular dynamicssimulations for Arg31Ala and Asn47Ala mutants of NRG-1β in the complexwith ErbB3 and ErbB4 were performed. The RMSD values of Cα a atomsbetween the average structure of the trajectories of the mutantcomplexes and the starting model structure are small, fluctuatingbetween 1.9 and 2.1 Å. ΔGsubtotal values recalculated based on the newtrajectories of the mutant complexes, as listed in Table 5, are almostidentical to the results derived from the modified trajectories ofwild-type complexes. The differences between ΔGsubtotal valuescalculated using two methods are less than 1 kcal-mol-1 (Table 5). Thisindicates that global conformations of NRG-1β/ErbB3 and NRG-1β/ErbB4 donot change dramatically after single alanine mutation.

TABLE 5 Comparison of the components of the binding free energy of theArg31Ala and Asn47Ala complex with ErbB receptors calculated from themodified trajectory of wild type and from the trajectories collected forthe NRG-1β Arg31Ala and Asn47Ala mutants. Arg31Ala modified Trajectoryof Asn47Ala modified Trajectory of trajectory of the the Arg31Alatrajectory of the the Asn47Ala wild type^(a) mutant^(b) wild type^(a)mutant^(b) Mean Std Mean Std Mean Std Mean Std ErbB3 ContributionΔE_(ele) −375.84 25.66 −481.47 28.15 −443.90 27.79 −547.58 15.65ΔE_(vdw) −141.02 7.16 −143.31 7.28 −140.22 6.72 −151.41 5.89 ΔE_(gas)−516.86 25.50 −624.78 27.24 −584.12 27.45 −698.99 15.56 ΔG_(nonpolar)−7.80 0.39 −8.51 0.60 −7.99 0.50 −9.44 0.34 ΔG_(PB) 488.48 25.81 596.6728.29 550.12 26.29 646.72 15.97 ΔG_(sol) 480.68 25.65 586.16 28.12542.13 26.11 657.27 15.93 ΔG_(subtotal) −36.18 7.30 −36.62 7.55 −42.016.86 −41.71 7.82 ErbB4 Contribution ΔE_(ele) −341.13 27.04 −382.51 20.14−371.70 27.00 −476.51 9.18 ΔE_(vdm) −128.92 6.64 −151.00 6.14 −130.886.78 −139.35 5.93 ΔE_(gas) −470.04 26.18 −533.51 19.18 −502.58 26.43−615.88 18.92 ΔG_(nonpolar) −7.26 0.52 −8.46 1.29 −7.58 0.51 −8.09 0.41ΔG_(PB) 447.59 25.20 511.86 19.34 480.25 25.32 593.59 14.90 ΔG_(sol)440.33 25.08 503.40 19.65 472.67 25.19 585.51 14.80 ΔG_(subtotal) −29.719.14 −30.11 22.59 −29.91 10.24 −30.37 13.36 ^(a)Calculated for themodified trajectories of the wild type by computational alanine-scanningmutagenesis experiments. ^(b)Calculated for the separate trajectoriescollected for the NRG-1β mutants.7. Comparative Studies of the Receptor Binding Affinity and SignalTransduction Ability of Neuregulin and its Mutations

To study the receptor binding affinity of neuregulin and its mutations,ErbB2&ErbB3 or ErbB2&ErbB4 co-expressing COS7 are used. The cells aregrown to 80% confluence. In the afternoon, after the cells reach 80%confluence, the media is changed to serum free media. Then, after 24hours, the cells are harvested for receptor binding affinity assays.

Similarly, to study the signal transduction ability of neuregulin andits mutations, ErbB2&ErbB3 or ErbB2&ErbB4 co-expressing COS7 cells areused. The cells are grown to 80% confluence. In the afternoon, after thecells reach 80% confluence, the media is changed to serum free media.Then after 24 hours, different amounts of neuregulin or neuregulinvariants are added into separate wells containing the cells. After 10minutes, loading buffer is added to lyse the cells. The sample is thenharvested and loaded into a separate well of gel for electrophoresis andwestern blot analysis.

The results for neuregulin (the 177-237 amino acids fragment ofneuregulin1 β2) and neuregulin variants L3A and D43A (alaninesubstitutions for the 3^(rd) and 43^(rd) amino acids, respectively, ofthe neuregulin fragment) are shown in Table 6, Table 7, FIG. 9 and FIG.10. The data in Table 6 and Table 7 show that L3A and D43A have nearlythe same receptor binding affinity for ErbB2&ErbB3 co-expressing cellsas neuregulin (if the EC50 or KD value is higher, then the receptorbinding affinity is lower), while D43A has a much higher (Table 6) andL3A has a much lower (Table 7) receptor binding affinity for ErbB2&ErbB4co-expressing cells than neuregulin. Similarly, FIGS. 9 and 10 show thatL3A and D43A binding induces more AKT phosphorylation than neuregulin inErbB2&ErbB4 co-expressing cells, but less AKT phosphorylation thanneuregulin in ErbB2&ErbB3 co-expressing cells. These results show thatL3A and D43A binding can strongly activate AKT signaling pathway inErbB2&ErbB4 co-expressing cells, but only activate a little inErbB2&ErbB3 co-expressing cells. These results are significant becausecardiac cells primarily express ErbB2&ErbB4, and variants, such as D43Aand L3A, can therefore be used to treat cardiovascular diseases whilereducing the side effect of neuregulin binding other ErbB2&ErbB3expressing cells.

TABLE 6 Comparison of the receptor binding affinity of neuregulin andvariant D43A on COS7 cells expressing ErbB2/ErbB3 and ErbB2/ErbB4,respectively. EC50 (nM) COS7 cells COS7 cells (ErbB2&ErbB3 (ErbB2&ErbB4Name co-expression) co-expression) NRG 112.9 132.4 D43A 149.2 40.66 EC50is the concentration of ligands which can compete 50% of boundradiolabeled ligands off the receptor complex.

TABLE 7 Comparison of the receptor binding affinity of neuregulin andvariant L3A on COS7 cells expressing ErbB2/ErbB3 and ErbB2/ErbB4,respectively. KD (pM) COS7 cells COS7 cells (ErbB2&ErbB3 (ErbB2&ErbB4Name co-expression) co-expression) NRG 1407 281.6 L3A 1151 1060 KDequals ([ligand] × [receptor])/[ligand · receptor complex], it reflectsthe off rate of ligand from receptor. [ligand] stands for theconcentration of ligand.

Neuregulin double mutant 8A47A (simultaneous alanine substitutions forthe 8^(th) and 47^(th) amino acids of the neuregulin fragment) wereconstructed. The same method as above was used to study the signaltransduction ability of neuregulin and its double mutants. The resultsfor neuregulin (the 177-237 amino acids fragment of neuregulin1β2) andneuregulin double mutant 8A47A (simultaneous alanine substitutions forthe 8^(th) and 47^(th) amino acids of the neuregulin fragment) are shownin FIG. 11. FIG. 11 show that 8A47A binding induces much more AKTphosphorylation than neuregulin in ErbB2&ErbB4 co-expressing cells, butnearly same AKT phosphorylation as neuregulin in ErbB2&ErbB3co-expressing cells. The result show that 8A47A binding can stronglyactivate AKT signaling pathway in ErbB2&ErbB4 co-expressing cells, whileonly normally activate the pathway in ErbB2&ErbB3 co-expressing cells.The result is very important because cardiac cells primarily expressErbB2&ErbB4, so 8A47A can be used to treat cardiovascular diseases whilemaintaining the side effect of neuregulin binding other ErbB2&ErbB3expressing cells.

CONCLUSIONS

Homology modeling, molecular dynamics and free energy calculationmethods were used to study interactions between NRG-1β and itsreceptors, ErbB3 and ErbB4. Binding features of NRG-1β to ErbB3 andErbB4 were addressed using molecular dynamics (MD) simulations. Thebinding free energies between the ligand and receptor were calculated bythe MM-PBSA method. MD simulations revealed that a number ofstructurally important residues of NRG-1β, such as Leu33, Arg44, Tyr48and Met 50, are retained for binding to the receptors comparison withcorresponding residues of EGF to its receptor. The free energycalculations between the ligand and receptors helped to dissect theorigin of binding affinities of NRG-1β to the receptors.

Moreover, the computational alanine-scanning method was used to map thecontribution of each residue at the interaction interfaces to thebinding affinity, and to validate the constructed models of theligand-receptor complexes by examining the functions of individualresidues at the binding sites. The computational alanine-scanningresults identified several important interaction residue pairs, forexample in the bindings of NRG-1β to ErbB3 and ErbB4, which shows goodagreement with experimental mutagenesis results. This indicates that thecurrent structural models of NRG-1β/ErbB3 and NRG-1β/ErbB4 complexes arereliable and are valuable for selecting desirable mutations on NRG-1β toincrease the binding affinity and selectivity to the receptor anddiscovering new therapeutic agents for the treatment of heart failure,and neuregulin mutations L3A and D43A strongly support this idea.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

What is claimed is:
 1. An isolated polypeptide variant of neuregulin-1βcomprising amino acid sequence shown in SEQ ID NO:1, wherein thepolypeptide variant comprises two different amino acids than that in SEQID NO:1, wherein the polypeptide variant has an enhanced bindingaffinity to ErbB4 compared to polypeptide of SEQ ID NO:1, and wherein atresidue 47 the first of said two different amino acids is A, C, D, E, F,G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y, and at residue 8 the secondof said two different amino acids is A.
 2. The polypeptide variant ofclaim 1, wherein the amino acid at residue 47 is A.
 3. The polypeptidevariant of claim 1, wherein the amino acid at residue 47 is C.
 4. Thepolypeptide variant of claim 1, wherein the amino acid at residue 47 isD.
 5. The polypeptide variant of claim 1, wherein the amino acid atresidue 47 is E.
 6. The polypeptide variant of claim 1, wherein theamino acid at residue 47 is F.
 7. The polypeptide variant of claim 1,wherein the amino acid at residue 47 is G.
 8. The polypeptide variant ofclaim 1, wherein the amino acid at residue 47 is H.
 9. The polypeptidevariant of claim 1, wherein the amino acid at residue 47 is I.
 10. Thepolypeptide variant of claim 1, wherein the amino acid at residue 47 isK.
 11. The polypeptide variant of claim 1, wherein the amino acid atresidue 47 is L.
 12. The polypeptide variant of claim 1, wherein theamino acid at residue 47 is M.
 13. The polypeptide variant of claim 1,wherein the amino acid at residue 47 is P.
 14. The polypeptide variantof claim 1, wherein the amino acid at residue 47 is Q.
 15. Thepolypeptide variant of claim 1, wherein the amino acid at residue 47 isR.
 16. The polypeptide variant of claim 1, wherein the amino acid atresidue 47 is S.
 17. The polypeptide variant of claim 1, wherein theamino acid at residue 47 is T.
 18. The polypeptide variant of claim 1,wherein the amino acid at residue 47 is V.
 19. The polypeptide variantof claim 1, wherein the amino acid at residue 47 is W.
 20. Thepolypeptide variant of claim 1, wherein the amino acid at residue 47 isY.
 21. A composition comprising the polypeptide variant of claim
 1. 22.The composition of claim 21, wherein the composition further comprises apharmaceutically acceptable excipient.
 23. The composition of claim 21,wherein the polypeptide variant comprises at residue 47 the amino acidA.
 24. The composition of claim 21, wherein the polypeptide variantcomprises at residue 47 the amino acid C.
 25. The composition of claim21, wherein the polypeptide variant comprises at residue 47 the aminoacid D.
 26. The composition of claim 21, wherein the polypeptide variantcomprises at residue 47 the amino acid E.
 27. The composition of claim21, wherein the polypeptide variant comprises at residue 47 the aminoacid F.
 28. The composition of claim 21, wherein the polypeptide variantcomprises at residue 47 the amino acid G.
 29. The composition of claim21, wherein the polypeptide variant comprises at residue 47 the aminoacid H.
 30. The composition of claim 21, wherein the polypeptide variantcomprises at residue 47 the amino acid I.
 31. The composition of claim21, wherein the polypeptide variant comprises at residue 47 the aminoacid K.
 32. The composition of claim 21, wherein the polypeptide variantcomprises at residue 47 the amino acid L.
 33. The composition of claim21, wherein the polypeptide variant comprises at residue 47 the aminoacid M.
 34. The composition of claim 21, wherein the polypeptide variantcomprises at residue 47 the amino acid P.
 35. The composition of claim21, wherein the polypeptide variant comprises at residue 47 the aminoacid Q.
 36. The composition of claim 21, wherein the polypeptide variantcomprises at residue 47 the amino acid R.
 37. The composition of claim21, wherein the polypeptide variant comprises at residue 47 the aminoacid S.
 38. The composition of claim 21, wherein the polypeptide variantcomprises at residue 47 the amino acid T.
 39. The composition of claim21, wherein the polypeptide variant comprises at residue 47 the aminoacid V.
 40. The composition of claim 21, wherein the polypeptide variantcomprises at residue 47 the amino acid W.
 41. The composition of claim21, wherein the polypeptide variant comprises at residue 47 the aminoacid Y.
 42. A kit comprising the polypeptide variant of
 1. 43. The kitof claim 42, wherein the polypeptide variant comprises at residue 47 theamino acid A.
 44. The kit of claim 42, wherein the polypeptide variantcomprises at residue 47 the amino acid C.
 45. The kit of claim 42,wherein the polypeptide variant comprises at residue 47 the amino acidD.
 46. The kit of claim 42, wherein the polypeptide variant comprises atresidue 47 the amino acid E.
 47. The kit of claim 42, wherein thepolypeptide variant comprises at residue 47 the amino acid F.
 48. Thekit of claim 42, wherein the polypeptide variant comprises at residue 47the amino acid G.
 49. The kit of claim 42, wherein the polypeptidevariant comprises at residue 47 the amino acid H.
 50. The kit of claim42, wherein the polypeptide variant comprises at residue 47 the aminoacid I.
 51. The kit of claim 42, wherein the polypeptide variantcomprises at residue 47 the amino acid K.
 52. The kit of claim 42,wherein the polypeptide variant comprises at residue 47 the amino acidL.
 53. The kit of claim 42, wherein the polypeptide variant comprises atresidue 47 the amino acid M.
 54. The kit of claim 42, wherein thepolypeptide variant comprises at residue 47 the amino acid P.
 55. Thekit of claim 42, wherein the polypeptide variant comprises at residue 47the amino acid Q.
 56. The kit of claim 42, wherein the polypeptidevariant comprises at residue 47 the amino acid R.
 57. The kit of claim42, wherein the polypeptide variant comprises at residue 47 the aminoacid S.
 58. The kit of claim 42, wherein the polypeptide variantcomprises at residue 47 the amino acid T.
 59. The kit of claim 42,wherein the polypeptide variant comprises at residue 47 the amino acidV.
 60. The kit of claim 42, wherein the polypeptide variant comprises atresidue 47 the amino acid W.
 61. The kit of claim 42, wherein thepolypeptide variant comprises at residue 47 the amino acid Y.
 62. Thepolypeptide variant of claim 1, wherein the polypeptide variant consistsof the amino acid sequence shown in SEQ ID NO:1 and wherein the aminoacid at residue 47 is A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V,W, or Y, and the amino acid at residue 8 is A.
 63. The polypeptidevariant of claim 62, wherein the amino acid at residue 47 is A.
 64. Thepolypeptide variant of claim 62, wherein the amino acid at residue 47 isC.
 65. The polypeptide variant of claim 62, wherein the amino acid atresidue 47 is D.
 66. The polypeptide variant of claim 62, wherein theamino acid at residue 47 is E.
 67. The polypeptide variant of claim 62,wherein the amino acid at residue 47 is F.
 68. The polypeptide variantof claim 62, wherein the amino acid at residue 47 is G.
 69. Thepolypeptide variant of claim 62, wherein the amino acid at residue 47 isH.
 70. The polypeptide variant of claim 62, wherein the amino acid atresidue 47 is I.
 71. The polypeptide variant of claim 62, wherein theamino acid at residue 47 is K.
 72. The polypeptide variant of claim 62,wherein the amino acid at residue 47 is L.
 73. The polypeptide variantof claim 62, wherein the amino acid at residue 47 is M.
 74. Thepolypeptide variant of claim 62, wherein the amino acid at residue 47 isP.
 75. The polypeptide variant of claim 62, wherein the amino acid atresidue 47 is Q.
 76. The polypeptide variant of claim 62, wherein theamino acid at residue 47 is R.
 77. The polypeptide variant of claim 62,wherein the amino acid at residue 47 is S.
 78. The polypeptide variantof claim 62, wherein the amino acid at residue 47 is T.
 79. Thepolypeptide variant of claim 62, wherein the amino acid at residue 47 isV.
 80. The polypeptide variant of claim 62, wherein the amino acid atresidue 47 is W.
 81. The polypeptide variant of claim 62, wherein theamino acid at residue 47 is Y.
 82. A composition comprising thepolypeptide variant of claim
 62. 83. The composition of claim 82,wherein the composition further comprises a pharmaceutically acceptableexcipient.
 84. A kit comprising the polypeptide variant of 62.